Plants Having Enhanced Yield-Related Traits and a Method for Making the Same
The present invention provides a method for enhancing various economically important yield-related traits in plants by modulating expression in a plant of a nucleic acid encoding a PATL (PATELLIN) polypeptide, a PRP38 (Precursor RNA Processing factor 38) polypeptide, an ADA2 (Adaptor 2) polypeptide, a GATA-like polypeptide, or a WD40 repeat (WDR) 23-like polypeptide. The present invention also concerns plants having modulated expression of a nucleic acid encoding a PATL polypeptide, a PRP38 polypeptide, an ADA2 polypeptide, a GATA-like polypeptide, or a WDR23-like polypeptide, which plants have improved yield-related traits relative to corresponding wild type plants or other control plants. The invention also provides nucleic acids and constructs useful in the methods of the invention.
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This application is a divisional of patent application Ser. No. 12/744,503 filed May 25, 2010, which is a national stage application (under 35 U.S.C. §371) of PCT/EP2008/066237, filed Nov. 26, 2008, which claims benefit of European application 07121546.1, filed Nov. 26, 2007, European Application 07121565.1, filed Nov. 26, 2007, European Application 07122488.5, filed Dec. 6, 2007, European Application 07122911.6, filed Dec. 11, 2007, European Application 07122998.3, filed Dec. 12, 2007, U.S. Provisional Application 61/013,649, filed Dec. 14, 2007, U.S. Provisional Application 61/013,648, filed Dec. 14, 2007, U.S. Provisional Application 61/014,619, filed Dec. 18, 2007, U.S. Provisional Application 61/014,757 filed, Dec. 19, 2007, U.S. Provisional Application 61/027,053, filed Feb. 8, 2008, European Application 08166636.4, filed on Oct. 15, 2008, and U.S. Provisional Application 61/106,989, filed Oct. 21, 2008. The entire content of each aforementioned application is hereby incorporated by reference in its entirety.
SUBMISSION OF SEQUENCE LISTINGThe Sequence Listing associated with this application is filed in electronic format via EFS-Web and hereby incorporated by reference into the specification in its entirety. The name of the text file containing the Sequence Listing is Sequence_Listing—13311—00093. The size of the text file is 703 KB, and the text file was created on Feb. 7, 2014.
The present invention relates generally to the field of molecular biology and concerns a method for enhancing yield related traits by modulating expression in a plant of a nucleic acid encoding a PATL (PATELLIN) polypeptide, or a PRP38 (Precursor RNA Processing factor 38), or a GATA-like polypeptide, or an ADA2 (Transcriptional Adaptor 2) polypeptide, or a WDR23-like polypeptide. The present invention also concerns plants having modulated expression of a nucleic acid encoding a PATL polypeptide, or PRP38, or a GATA-like, or an ADA2 polypeptide, or a WDR23-like which plants have enhanced yield-related traits relative to corresponding wild type plants or other control plants. The invention also provides hitherto unknown PATL, or PRP38, or GATA-like, or ADA2, or WDR23-like nucleic acids and constructs useful in the methods of the invention.
In one embodiment, the present invention also concerns a method for improving plant growth characteristics by modulating expression in a plant of a nucleic acid encoding a GATA-like polypeptide. The present invention also concerns plants having modulated expression of a nucleic acid encoding a GATA-like polypeptide, which plants have improved growth characteristics relative to corresponding wild type plants or other control plants. The invention also provides constructs useful in the methods of the invention.
In another embodiment, the present invention concerns a method for increasing various plant yield-related traits by increasing expression in a plant of a nucleic acid sequence encoding a WD40 repeat (WDR) 23-like polypeptide. The present invention also concerns plants having increased expression of a nucleic acid sequence encoding a WDR23-like polypeptide, which plants have increased yield-related traits relative to control plants. The invention additionally relates to specific nucleic acid sequences encoding the aforementioned polypeptides having the aforementioned plant yield increasing activity, nucleic acid constructs, vectors and plants containing said nucleic acid sequences.
The ever-increasing world population and the dwindling supply of arable land available for agriculture fuels research towards increasing the efficiency of agriculture. Conventional means for crop and horticultural improvements utilise selective breeding techniques to identify plants having desirable characteristics. However, such selective breeding techniques have several drawbacks, namely that these techniques are typically labour intensive and result in plants that often contain heterogeneous genetic components that may not always result in the desirable trait being passed on from parent plants. Advances in molecular biology have allowed mankind to modify the germplasm of animals and plants. Genetic engineering of plants entails the isolation and manipulation of genetic material (typically in the form of DNA or RNA) and the subsequent introduction of that genetic material into a plant. Such technology has the capacity to deliver crops or plants having various improved economic, agronomic or horticultural traits.
A trait of particular economic interest is increased yield. Yield is normally defined as the measurable produce of economic value from a crop. This may be defined in terms of quantity and/or quality. Yield is directly dependent on several factors, for example, the number and size of the organs, plant architecture (for example, the number of branches), seed production, leaf senescence and more. Root development, nutrient uptake, stress tolerance and early vigour may also be important factors in determining yield. Optimizing the abovementioned factors may therefore contribute to increasing crop yield.
Seed yield is a particularly important trait, since the seeds of many plants are important for human and animal nutrition. Crops such as corn, rice, wheat, canola and soybean account for over half the total human caloric intake, whether through direct consumption of the seeds themselves or through consumption of meat products raised on processed seeds. They are also a source of sugars, oils and many kinds of metabolites used in industrial processes. Seeds contain an embryo (the source of new shoots and roots) and an endosperm (the source of nutrients for embryo growth during germination and during early growth of seedlings). The development of a seed involves many genes, and requires the transfer of metabolites from the roots, leaves and stems into the growing seed. The endosperm, in particular, assimilates the metabolic precursors of carbohydrates, oils and proteins and synthesizes them into storage macromolecules to fill out the grain.
Plant biomass is yield for forage crops like alfalfa, silage corn and hay. Many proxies for yield have been used in grain crops. Chief amongst these are estimates of plant size. Plant size can be measured in many ways depending on species and developmental stage, but include total plant dry weight, above-ground dry weight, above-ground fresh weight, leaf area, stem volume, plant height, rosette diameter, leaf length, root length, root mass, tiller number and leaf number. Many species maintain a conservative ratio between the size of different parts of the plant at a given developmental stage. These allometric relationships are used to extrapolate from one of these measures of size to another (e.g. Tittonell et al 2005 Agric Ecosys & Environ 105: 213). Plant size at an early developmental stage will typically correlate with plant size later in development. A larger plant with a greater leaf area can typically absorb more light and carbon dioxide than a smaller plant and therefore will likely gain a greater weight during the same period (Fasoula & Tollenaar 2005 Maydica 50:39). This is in addition to the potential continuation of the micro-environmental or genetic advantage that the plant had to achieve the larger size initially. There is a strong genetic component to plant size and growth rate (e.g. ter Steege et al 2005 Plant Physiology 139:1078), and so for a range of diverse genotypes plant size under one environmental condition is likely to correlate with size under another (Hittalmani et al 2003 Theoretical Applied Genetics 107:679). In this way a standard environment is used as a proxy for the diverse and dynamic environments encountered at different locations and times by crops in the field.
Another important trait for many crops is early vigour. Improving early vigour is an important objective of modern rice breeding programs in both temperate and tropical rice cultivars. Long roots are important for proper soil anchorage in water-seeded rice. Where rice is sown directly into flooded fields, and where plants must emerge rapidly through water, longer shoots are associated with vigour. Where drill-seeding is practiced, longer mesocotyls and coleoptiles are important for good seedling emergence. The ability to engineer early vigour into plants would be of great importance in agriculture. For example, poor early vigour has been a limitation to the introduction of maize (Zea mays L.) hybrids based on Corn Belt germplasm in the European Atlantic.
Harvest index, the ratio of seed yield to aboveground dry weight, is relatively stable under many environmental conditions and so a robust correlation between plant size and grain yield can often be obtained (e.g. Rebetzke et al 2002 Crop Science 42:739). These processes are intrinsically linked because the majority of grain biomass is dependent on current or stored photosynthetic productivity by the leaves and stem of the plant (Gardener et al 1985 Physiology of Crop Plants. Iowa State University Press, pp 68-73). Therefore, selecting for plant size, even at early stages of development, has been used as an indicator for future potential yield (e.g. Tittonell et al 2005 Agric Ecosys & Environ 105: 213). When testing for the impact of genetic differences on stress tolerance, the ability to standardize soil properties, temperature, water and nutrient availability and light intensity is an intrinsic advantage of greenhouse or plant growth chamber environments compared to the field. However, artificial limitations on yield due to poor pollination due to the absence of wind or insects, or insufficient space for mature root or canopy growth, can restrict the use of these controlled environments for testing yield differences. Therefore, measurements of plant size in early development, under standardized conditions in a growth chamber or greenhouse, are standard practices to provide indication of potential genetic yield advantages.
Another trait of importance is that of improved abiotic stress tolerance. Abiotic stress is a primary cause of crop loss worldwide, reducing average yields for most major crop plants by more than 50% (Wang et al. (2003) Planta 218: 1-14). Abiotic stresses may be caused by drought, salinity, extremes of temperature, chemical toxicity, excess or deficiency of nutrients (macroelements and/or microelements), radiation and oxidative stress. The ability to increase plant tolerance to abiotic stress would be of great economic advantage to farmers worldwide and would allow for the cultivation of crops during adverse conditions and in territories where cultivation of crops may not otherwise be possible.
Crop yield may therefore be increased by optimising one of the above-mentioned factors.
Depending on the end use, the modification of certain yield traits may be favoured over others. For example for applications such as forage or wood production, or bio-fuel resource, an increase in the vegetative parts of a plant may be desirable, and for applications such as flour, starch or oil production, an increase in seed parameters may be particularly desirable. Even amongst the seed parameters, some may be favoured over others, depending on the application. Various mechanisms may contribute to increasing seed yield, whether that is in the form of increased seed size or increased seed number.
One approach to increasing yield-related traits (seed yield and/or biomass) in plants may be through modification of the inherent growth mechanisms of a plant, such as the cell cycle or various signalling pathways involved in plant growth or in defense mechanisms.
It has now been found that various yield-related traits may be enhanced in plants by modulating expression in a plant of a nucleic acid encoding a PATL (Patellin) polypeptide, or a PRP38 (Precursor RNA Processing factor 38), or an ADA2 (Transcriptional Adaptor 2) polypeptide, or a WD40 repeat (WDR) 23-like polypeptide.
It has furthermore been found that various growth characteristics may be improved in plants by modulating expression in a plant of a nucleic acid encoding a GATA-like (Protein Of Interest) in a plant.
The increased yield-related traits comprise one or more of: increased total seed yield per plant, increased seed filling rate, increased number of filled seeds, increased harvest index, or increased thousand kernel weight.
Lipids are substances soluble in non-polar solvents, such as chloroform or ether. Lipids are essential components in living organisms. Lipid such as glycolipids, phospholipids, and cholesterol are key structural components of cell membranes, and triglycerides are the biological energy-source molecules. Phosphatidylinositide (PtdIns) and Phosphatidylcholine (PtdCho) are examples of phospholipids. Interactions between lipids and proteins function in targeting proteins and glycolipids involved in a variety of processes, such as cell signaling and cell proliferation, to specific membrane and intracellular locations. Various proteins are associated with the biosynthesis, transport, and uptake of lipids. In addition, key proteins involved in signal transduction and protein targeting have lipid-derived groups added to them post-translationally (Stryer, L. (1995) Biochemistry, W.H. Freeman and Co., New York N.Y., pp. 264-267, 934).
Phosphatidylinositol/phosphatidylcholine transfer proteins (PIPTs) are ubiquitous proteins involved in coordinate regulation of PtdIns and PtdCho metabolism by facilitating the transfer among the different membrane compartments of eukatyotic cells. In yeast, the major PIPT is the SEC14 protein which is essential for secretion from the trans-Golgi network (Bankatis et al. 1989, J. Cell Biol. 108:1271-81). Similar proteins are found in plants and other higher organisms. In plants, several SEC14 homologs have been described to have around 25% amino acid sequence similarity to the yeast S. cerevisiae SEC14 protein and in some instances the putative PtdIns/PtdCho transfer function has been experimentally verified by complementation of the sec14-1 temperature-sensitive yeast mutant (Jouannic et al. 1998). Plant SEC14 proteins reportedly play a role in diverse biological processes such as cytokinesis, hyperosmosis stress-induced signalling pathway, vesicle trafficking between various membranes or membrane biogenesis during nodulation (Allen-Baume et al. 2002; FEBs Letters 531: 74-80; Peterman et al. 2004. Plant Phys. 136: 3080-3094; Monks et al. 2001, Plant Cell 13:1205-19; Kapranov et al. 2001 Plant Cell 13:1369-1382).
In Arabidopsis thaliana the Patellin1 (PATL1) protein, a member of the SEC14 homolog protein family, has been reported to localize to the cell plate during cytokinesis and shown to have PtdIns binding activity. PATL1 is a member of a small family of six proteins in Arabidopsis characterized by the presence of two conserved domains, a SEC14 and a GOLD (Golgi dynamics) domain.
The SEC14 domain is found in the SEC14 protein of yeast and in in RhoGAPs, RhoGEFs and the RasGAP, neurofibromin (NF1). It is also found in C-terminal of various retinaldehyde/retinal-binding proteins (CRAL-BP) that may be functional components of the visual cycle and in the Trio protein, a multifunctional factor that integrates and amplifies signals involved in actin remodelling. SEC14 is sometimes refer to as CRAL_TRIO domain. SEC14 domain is involved in lipid binding (Sha and Luo. 1999. Biochim Biophys Acta. 1441: 268-77).
The GOLD domain is a protein module found in several eukaryotic Golgi and lipid-traffic proteins. It is typically between 90 and 150 amino acids long. Most of the size difference observed in the GOLD-domain superfamily is traceable to a single large low-complexity insert that is seen in some versions of the domain. GOLD domains occur in proteins that may interact with membranes or which may have a role in the interaction of various proteins with cytoskeletal filaments such as animal SEC14 proteins or the yeast oxysterol-binding protein homolog 3 (OSH3). The GOLD domain is predicted to mediate diverse protein-protein interactions (Anantharaman et al. 2002. Genome Biol. 3).
The patent application WO 2004/090141 describes a partial PATL protein involved in stress tolerance.
The synthesis and function of messenger RNA (mRNA) in a cell requires a series of events including transcription, processing, transport, translation and degradation. RNA processing refers to events modifying RNA posttranscriptionally. In eukaryotic organisms the majority of the nascent pre-mRNA contains introns, which are spliced out resulting in the precise ligation of exons to produce a mature mRNA, which is the RNA form used by the ribosomes to translate into a protein. Posttranscriptional modification of RNAs also includes capping at the 5′ end and polyadenylation at the 3′ end, which affects stability and the efficiency of translation.
The relationship between mRNA translation and turnover is critical to the regulation of gene expression and to the correct functioning of the cell. In eukaryotic organisms, several tens of proteins are involved in RNA metabolism. An example of such a protein is the pre-mRNA splicing factor PRP38.
Yeast PRP38 was identified in a genetic screen of temperature-sensitive mutants of Saccharomyces cerevisiae defective in pre-mRNA splicing (Blanton et al. 1992, Mol. Cel. Biol. 12, 3939-3947). The excision of intervening sequences from eukaryotic pre-mRNA transcripts occurs in the nucleus on a large, complex structure termed the spliceosome. The spliceosome directs intron removal by a two-step mechanism comprising (i) cleavage at the 5′ splice site and ligation of the 5′-terminal nucleotide of the intron to an adenosine near the 3′ end on the intron and (ii) cleavage at the 3′ splice site and exon ligation. Assembly of the spliceosome progresses by the sequential addition of the U1, U2 U4/U6 and U5 snRNPs (Small Ribonuclear Proteins U1-U6). Late in the assembly process the U4 disassociates from the spliceosome. The catalytic events of splicing leading to intron removal in pre-mRNA molecules are believed to occur subsequent to U4 dissociation. In yeast, PRP38 is reportedly dispensable for spliceosome assembly but required for conformational changes which lead to catalytic activation of the spliceosome (Xie et al. (1998) EMBO Journal Vol. 17 pp. 2938-2946).
In Arabidopsis thaliana, PRP38 protein (AtPRP38) was named SRL1 (SR like 1), after its sequence similarity with SR proteins (Forment et al. Plant J. 2002 June; 30(5):511-9. However PRP38 is structurally distinct from other SR proteins. Typically SR proteins comprise an RRM domain (RNA binding domain) and an RS domain (Kalyna and Barta Biochem Soc Trans. 2004 32:561-4.), while AtPRP38 comprises the RS domain, but lacks the RRM domain.
WO 01/81599 describes methods to improve stress tolerance in yeast and plants using nucleic acids encoding several SR proteins. SRL1 as well as other proteins functioning in pre-mRNA splicing have been found to play an important role in plant responses to abiotic stresses (Lee et al. 2006 Plant Cell. July; 18(7):1736-49).
The GATA family forms one of the major families in Cys2/Cys2-type zinc finger transcription factor in eukaryotes (found e.g. in cellular slime mold, plants, fungi, nematodes, insects, echinoderms, vertebrates) and contain either 1 or 2 highly conserved zinc finger DNA binding domains. The DNA-binding consensus is CX2CX17-20CX2C and contains the zinc atom coordinated by the conserved 4 cysteines (Omichinski et al., 1993; Reyes et al., 2004). This domain is typically followed by a basic region. It characteristically binds DNA at the GATA recognition sequence (A/T)GATA(A/G) (Martin and Orkin, 1990; Omichinski et al., 2003). Animal GATAs typically contain 17 residues between second and third cystein, whereas in fungi this can be 17-18 (rarely 19 or 20). Plants have 18 or 20 residues between the second and third cystein.
The original GATA1 was identified as a transcription factor required to promote the expression of globin genes in humans (Pevny et al., 1991). The first plant GATA (NTL1) was isolated form tobacco as a plant homologue of the Neurospora crassa transcription factor NIT2, a protein that activates the expression of the genes for nitrogen-metabolic enzymes during nitrogen-limiting conditions. NTL1 contains 18 spacings between the second and the third cysteine (CX2CX18CX2C) (Daniel-Vedele and Caboche, 1997). The genomes of Arabidopsis thaliana and Oryza sativa present 29 and 28 loci respectively that encode for putative GATA transcription factors (CX2CX18or20CX2C) (Reyes et al., 2004): Arabidopsis only has proteins with one GATA domain, while Oryza sativa has two proteins containing two and one protein containing three GATA domains. The rice transcription factors database (DRTF, Gao et al. Bioinformatics 2006, 22, 1286-1287) claim 28 GATAs in Indica and 23 in Japonica. The GATA family of transcription factors in rice and Arabidopsis can be divided into 7 subfamilies, some of these represented in both species but others are exclusive for one of them.
Ectopic expression of a GATA-like protein (HAN) under control of the strong constitutive 35S promoter seriously affected plant viability and development (Zhao et al. Plant Cell 16, 2586-2600, 2004). The transformed plants were dwarfed with abnormally formed leaves and smaller and malformed inflorescences. US2007/0250956 discloses OsGATA11 and its use for increasing seed yield.
The regulation of transcription in plants underlies many biological processes such as adaptation to environment and regulation of metabolic and physiological balances. Therefore altering gene transcription in a plant may result in profound modifications of plant growth and development. This property may be used to improve performance of crop plants. There are hundreds of genes controlling gene expression and only a few of them have been shown to have a beneficial effect on traits of interest to agriculture industry, when its expression is modulated in the plan (Vinocur and Altman, 2005, Current Opinion in Biotechnology 16,123-132; Gutterson and Reuber 2004. Current Opinion in Plant Biology, 7, 465-471). The availability to modulate gene expression in order to enhance yield traits would contribute improve crop performance and ultimately benefit agriculture based industries.
In eukaryotes, gene expression is coupled to chromatin modifications resulting in promoters becoming more accessible to RNA Polymerase II and other components of the transcriptional apparatus. Two main classes of protein complexes influence chromatin dynamics. One class comprises ATP-dependent chromatin-remodeling machines (SWI/SNF related complexes) and a second class comprises complexes that modify histone proteins. One such histone modification, mediated by histone acetyltransferases (HATs), may be by acetylacion of specific Lysine residues present in the N-terminus of core histones (Narlikar et al. 2002 Cell 108, 475-487). One of the prototypical histone acetyltransferases that functions as a transcriptional coactivator is known as GcnS. In yeast, GcnS forms distinct protein complexes known to comprise different adaptor proteins such as ADA, SAGA, SALSA and SLIK [18-20]. Homologous complexes have been identified in higher eukaryotes including plants. In Arabidopsis thaliana two paralogous genes encoding ADA2 proteins have been described (Stockinger et al. 2001 Nucleic acid research 29, 1524-1533).
General transcription factors recruit RNA polymerase II to the TATA box of promoters while transcriptional activators bind to specific DNA sequences called the upstream activating sites (UAS). General transcription factors may interact directly with the transcriptional activator or through the adaptor proteins. Typically adaptor proteins interact with the transcription activator via the activation domain though interactions via the DNA biding domain have also been described (Mao et al. 2006. Biochimica et Biophysica Acta 1759 69-79).
In Arabidopsis thaliana ADA2 proteins reportedly enhance the ability of GCN5 to acetylate histones in vitro and modulate the substrates specificity of GCN5. Moreover, GCN5 can acetylate the ADA2 proteins at a motif unique to the plant homologs and absent from fungal and animal homologs (Mao et al. 2006). T-DNA insertion mutations in ADA2b and GCN5 were found to have pleiotropic effects on plant growth and development, including dwarf size, aberrant root development, and short petals and stamens in flowers (Vlachonasios et al. 2003 The Plant Cell, Vol. 15, 626-638). Genes encoding transcriptional adaptor proteins originating from other plant species have also been disclosed (WO0003026).
The regulation of many different cellular processes requires the use of protein interaction domains to direct association of polypeptides with one another, and with phospholipids, small molecules, or nucleic acids. One such protein interaction domain is called the WD repeat (see review Smith et al. (1999) TIBS 24: 181-185). WD repeat containing proteins are largely represented in most organisms, for example, more than 300 are counted in H. sapiens, more than 140 in C. elegans, more than 390 in A. thaliana, and more than 90 in S. cereviseae. Although structurally related by the presence of WD repeats (between 4 and 16 copies in one polypeptide), these proteins have very diverse functions.
The WD repeat is loosely defined at the primary sequence level by a Gly-His (GH) dipeptide 10-20 residues N-terminal from a Trp-Asp (WD) dipeptide (located at the C-terminus of the motif), and is typically approximately 40 (but up to 60) residues in length (hence the “WD40” name). However, neither the GH dipeptide nor the WD dipeptide is absolutely conserved. Between GH and WD is a conserved core sequence that can be identified using algorithms available, for example, at InterPro, hosted by the European Bioinformatics Institute (EBI) in the UK. InterPro is a database of protein families, domains and functional sites in which identifiable features found in known proteins can be applied to unknown protein sequences.
WD40 proteins are predicted to form a beta propeller-like structure (a circular repeating structure), which contains three potential interacting surfaces: the top, the bottom, and the circumference. This expansive surface area allows a WD40 protein to form complexes reversibly with several proteins, thus coordinating sequential and/or simultaneous interactions involving several sets of protein.
Among the very important protein complexes involving WD40 proteins is a class of multiprotein ubiquitin E3 ligases of which Cullin 4 (CUL4) and damaged DNA binding protein 1 (DDB1) are the core proteins (Higa et al. (2007) Cell Division 2:5; Angers et al. (2006) Nature 443: 590-593; Higa et al. (2006) Nature Cell Biol 8(11): 1277-1283; He et al. (2006) Genes & Development 20: 2949-2954). This complex docks WD40 proteins as molecular adaptors for substrate recruiting mechanism, which substrate will subsequently be ubiquitinated and destroyed. These WD40 proteins form a subclass called DCAF (DDB1-CUL4A-associated factor), and comprise two conserved DxR motifs at the end of two consecutive WD40 repeats (He et al. (2006) supra).
One such WD40 protein, WDR23 (WD repeat 23; also called DCAF11) has significant primary sequence identity with a WD40 protein found in plants. This WD40 protein was first identified in Lithospermum erythrorhizon as clone 14B (LEC14B; NCBI accession number D83074).
In patent application WO2002/016655, SEQ ID NO: 2577 relates to an Arabidopsis thaliana nucleic acid sequence encoding a WDR23-like polypeptide, and is described a method of identifying a stress condition to which a plant cell has been exposed, using any one or more of SEQ ID NO: 1 through SEQ ID NO: 5379. In US patent application US20041034888, SEQ ID NO: 13,294 relates to an Arabidopsis thaliana nucleic acid sequence encoding a WDR23-like polypeptide, and is described a method of producing a plant having an improved property, using any one or more of SEQ ID NO: 1 through SEQ ID NO: 36,564.
Surprisingly, it has now been found that modulating expression of a nucleic acid encoding a PRP38 polypeptide gives plants having enhanced (or improved) yield-related traits, in particular increased yield relative to control plants, also in conditions other than osmotic stress caused by salt, drought, cold or freezing.
Also surprisingly, it has now been found that modulating (preferably increasing) expression of a nucleic acid encoding a GATA-like polypeptide gives plants having significantly increased Thousand Kernel Weight (TKW) relative to control plants, while other seed yield parameters such as number of seeds (filled seeds or total number of seeds) were not significantly increased.
Furthermore, surprisingly, it has now been found that modulating expression of a nucleic acid encoding an ADA2 polypeptide gives plants having enhanced yield-related traits, in particular increased yield relative to control plants.
Also surprisingly, it has now been found that increasing expression of a nucleic acid sequence encoding a WDR23-like polypeptide as defined herein gives plants having increased yield-related traits relative to control plants.
Furthermore, surprisingly, it has now been found that modulating expression of a nucleic acid encoding a PATL polypeptide gives plants having enhanced yield-related traits, in particular increased yield relative to control plants.
According one embodiment of the present invention, there is provided a method for improving (or enhancing) yield related traits in a plant relative to control plants, comprising modulating expression of a nucleic acid encoding a PATL polypeptide, or a PRP38, or an ADA2 polypeptide in a plant.
According to another embodiment, there is provided a method for increasing Thousand Kernel Weight (TKW) relative to control plants, comprising modulating (preferably increasing) expression of a nucleic acid encoding a GATA-like polypeptide in a plant.
According to another embodiment, there is provided a method for increasing yield-related traits in plants relative to control plants, comprising increasing expression of a nucleic acid sequence encoding a WDR23-like polypeptide as defined herein, in a plant. The increased yield-related traits comprise one or more of: increased total seed yield per plant, increased seed filling rate, increased number of filled seeds, increased harvest index, or increased thousand kernel weight.
DEFINITIONS Polypeptide(s)/Protein(s)The terms “polypeptide” and “protein” are used interchangeably herein and refer to amino acids in a polymeric form of any length, linked together by peptide bonds.
Polynucleotide(s)/Nucleic Acid(s)/Nucleic Acid Sequence(s)/Nucleotide Sequence(s)The terms “polynucleotide(s)”, “nucleic acid sequence(s)”, “nucleotide sequence(s)”, “nucleic acid(s)”, “nucleic acid molecule” are used interchangeably herein and refer to nucleotides, either ribonucleotides or deoxyribonucleotides or a combination of both, in a polymeric unbranched form of any length.
Control Plant(s)The choice of suitable control plants is a routine part of an experimental setup and may include corresponding wild type plants or corresponding plants without the gene of interest. The control plant is typically of the same plant species or even of the same variety as the plant to be assessed. The control plant may also be a nullizygote of the plant to be assessed. Nullizygotes are individuals missing the transgene by segregation. A “control plant” as used herein refers not only to whole plants, but also to plant parts, including seeds and seed parts.
Homologue(s)“Homologues” of a protein encompass peptides, oligopeptides, polypeptides, proteins and enzymes having amino acid substitutions, deletions and/or insertions relative to the unmodified protein in question and having similar biological and functional activity as the unmodified protein from which they are derived.
A deletion refers to removal of one or more amino acids from a protein.
An insertion refers to one or more amino acid residues being introduced into a predetermined site in a protein. Insertions may comprise N-terminal and/or C-terminal fusions as well as intra-sequence insertions of single or multiple amino acids. Generally, insertions within the amino acid sequence will be smaller than N- or C-terminal fusions, of the order of about 1 to 10 residues. Examples of N- or C-terminal fusion proteins or peptides include the binding domain or activation domain of a transcriptional activator as used in the yeast two-hybrid system, phage coat proteins, (histidine)-6-tag, glutathione S-transferase-tag, protein A, maltose-binding protein, dihydrofolate reductase, Tag.100 epitope, c-myc epitope, FLAG®-epitope, lacZ, CMP (calmodulin-binding peptide), HA epitope, protein C epitope and VSV epitope.
A substitution refers to replacement of amino acids of the protein with other amino acids having similar properties (such as similar hydrophobicity, hydrophilicity, antigenicity, propensity to form or break α-helical structures or β-sheet structures). Amino acid substitutions are typically of single residues, but may be clustered depending upon functional constraints placed upon the polypeptide; insertions will usually be of the order of about 1 to 10 amino acid residues. The amino acid substitutions are preferably conservative amino acid substitutions. Conservative substitution tables are well known in the art (see for example Creighton (1984) Proteins. W.H. Freeman and Company (Eds) and Table 1 below).
Amino acid substitutions, deletions and/or insertions may readily be made using peptide synthetic techniques well known in the art, such as solid phase peptide synthesis and the like, or by recombinant DNA manipulation. Methods for the manipulation of DNA sequences to produce substitution, insertion or deletion variants of a protein are well known in the art. For example, techniques for making substitution mutations at predetermined sites in DNA are well known to those skilled in the art and include M13 mutagenesis, T7-Gen in vitro mutagenesis (USB, Cleveland, Ohio), QuickChange Site Directed mutagenesis (Stratagene, San Diego, Calif.), PCR-mediated site-directed mutagenesis or other site-directed mutagenesis protocols.
Derivatives“Derivatives” include peptides, oligopeptides, polypeptides which may, compared to the amino acid sequence of the naturally-occurring form of the protein, such as the Patellin, or the Precursor RNA Processing factor 38, or a GATA-like polypeptide, or the Adaptor 2, or the WDR23-like polypeptide, comprise substitutions of amino acids with non-naturally occurring amino acid residues, or additions of non-naturally occurring amino acid residues. “Derivatives” of a protein also encompass peptides, oligopeptides, polypeptides which comprise naturally occurring altered (glycosylated, acylated, prenylated, phosphorylated, myristoylated, sulphated etc.) or non-naturally altered amino acid residues compared to the amino acid sequence of a naturally-occurring form of the polypeptide. A derivative may also comprise one or more non-amino acid substituents or additions compared to the amino acid sequence from which it is derived, for example a reporter molecule or other ligand, covalently or non-covalently bound to the amino acid sequence, such as a reporter molecule which is bound to facilitate its detection, and non-naturally occurring amino acid residues relative to the amino acid sequence of a naturally-occurring protein. Furthermore, “derivatives” also include fusions of the naturally-occurring form of the protein with tagging peptides such as FLAG, HIS6 or thioredoxin (for a review of tagging peptides, see Terpe, Appl. Microbiol. Biotechnol. 60, 523-533, 2003).
Ortholoque(s)/Paraloque(s)Orthologues and paralogues encompass evolutionary concepts used to describe the ancestral relationships of genes. Paralogues are genes within the same species that have originated through duplication of an ancestral gene; orthologues are genes from different organisms that have originated through speciation, and are also derived from a common ancestral gene.
DomainThe term “domain” refers to a set of amino acids conserved at specific positions along an alignment of sequences of evolutionarily related proteins. While amino acids at other positions can vary between homologues, amino acids that are highly conserved at specific positions indicate amino acids that are likely essential in the structure, stability or function of a protein. Identified by their high degree of conservation in aligned sequences of a family of protein homologues, they can be used as identifiers to determine if any polypeptide in question belongs to a previously identified polypeptide family.
Motif/Consensus sequence/Signature
The term “motif” or “consensus sequence” or “signature” refers to a short conserved region in the sequence of evolutionarily related proteins. Motifs are frequently highly conserved parts of domains, but may also include only part of the domain, or be located outside of conserved domain (if all of the amino acids of the motif fall outside of a defined domain).
HybridisationThe term “hybridisation” as defined herein is a process wherein substantially homologous complementary nucleotide sequences anneal to each other. The hybridisation process can occur entirely in solution, i.e. both complementary nucleic acids are in solution. The hybridisation process can also occur with one of the complementary nucleic acids immobilised to a matrix such as magnetic beads, Sepharose beads or any other resin. The hybridisation process can furthermore occur with one of the complementary nucleic acids immobilised to a solid support such as a nitro-cellulose or nylon membrane or immobilised by e.g. photolithography to, for example, a siliceous glass support (the latter known as nucleic acid arrays or microarrays or as nucleic acid chips). In order to allow hybridisation to occur, the nucleic acid molecules are generally thermally or chemically denatured to melt a double strand into two single strands and/or to remove hairpins or other secondary structures from single stranded nucleic acids.
The term “stringency” refers to the conditions under which a hybridisation takes place. The stringency of hybridisation is influenced by conditions such as temperature, salt concentration, ionic strength and hybridisation buffer composition. Generally, low stringency conditions are selected to be about 30° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. Medium stringency conditions are when the temperature is 20° C. below Tm, and high stringency conditions are when the temperature is 10° C. below Tm. High stringency hybridisation conditions are typically used for isolating hybridising sequences that have high sequence similarity to the target nucleic acid sequence. However, nucleic acids may deviate in sequence and still encode a substantially identical polypeptide, due to the degeneracy of the genetic code. Therefore medium stringency hybridisation conditions may sometimes be needed to identify such nucleic acid molecules.
The Tm is the temperature under defined ionic strength and pH, at which 50% of the target sequence hybridises to a perfectly matched probe. The Tm is dependent upon the solution conditions and the base composition and length of the probe. For example, longer sequences hybridise specifically at higher temperatures. The maximum rate of hybridisation is obtained from about 16° C. up to 32° C. below Tm. The presence of monovalent cations in the hybridisation solution reduce the electrostatic repulsion between the two nucleic acid strands thereby promoting hybrid formation; this effect is visible for sodium concentrations of up to 0.4M (for higher concentrations, this effect may be ignored). Formamide reduces the melting temperature of DNA-DNA and DNA-RNA duplexes with 0.6 to 0.7° C. for each percent formamide, and addition of 50% formamide allows hybridisation to be performed at 30 to 45° C., though the rate of hybridisation will be lowered. Base pair mismatches reduce the hybridisation rate and the thermal stability of the duplexes. On average and for large probes, the Tm decreases about 1° C. per % base mismatch. The Tm may be calculated using the following equations, depending on the types of hybrids:
1) DNA-DNA hybrids (Meinkoth and Wahl, Anal. Biochem., 138: 267-284, 1984):
Tm=81.5° C.+16.6×log10[Na+]a+0.41×%[G/Cb]−500×[Lc]−1−0.61×% formamide
2) DNA-RNA or RNA-RNA hybrids:
Tm=79.8+18.5(log10[Na+]a)+0.58(% G/Cb)+11.8(% G/Cb)2−820/Lc
3) oligo-DNA or oligo-RNAd hybrids:
For <20 nucleotides: Tm=2(ln)
For 20-35 nucleotides: Tm=22+1.46(ln)
a or for other monovalent cation, but only accurate in the 0.01-0.4 M range.
b only accurate for % GC in the 30% to 75% range.
c L=length of duplex in base pairs.
d oligo, oligonucleotide; ln, =effective length of primer=2×(no. of G/C)+(no. of NT).
Non-specific binding may be controlled using any one of a number of known techniques such as, for example, blocking the membrane with protein containing solutions, additions of heterologous RNA, DNA, and SDS to the hybridisation buffer, and treatment with Rnase. For non-homologous probes, a series of hybridizations may be performed by varying one of (i) progressively lowering the annealing temperature (for example from 68° C. to 42° C.) or (ii) progressively lowering the formamide concentration (for example from 50% to 0%). The skilled artisan is aware of various parameters which may be altered during hybridisation and which will either maintain or change the stringency conditions.
Besides the hybridisation conditions, specificity of hybridisation typically also depends on the function of post-hybridisation washes. To remove background resulting from non-specific hybridisation, samples are washed with dilute salt solutions. Critical factors of such washes include the ionic strength and temperature of the final wash solution: the lower the salt concentration and the higher the wash temperature, the higher the stringency of the wash.
Wash conditions are typically performed at or below hybridisation stringency. A positive hybridisation gives a signal that is at least twice of that of the background. Generally, suitable stringent conditions for nucleic acid hybridisation assays or gene amplification detection procedures are as set forth above. More or less stringent conditions may also be selected. The skilled artisan is aware of various parameters which may be altered during washing and which will either maintain or change the stringency conditions.
For example, typical high stringency hybridisation conditions for DNA hybrids longer than 50 nucleotides encompass hybridisation at 65° C. in 1×SSC or at 42° C. in 1×SSC and 50% formamide, followed by washing at 65° C. in 0.3×SSC. Examples of medium stringency hybridisation conditions for DNA hybrids longer than 50 nucleotides encompass hybridisation at 50° C. in 4×SSC or at 40° C. in 6×SSC and 50% formamide, followed by washing at 50° C. in 2×SSC. The length of the hybrid is the anticipated length for the hybridising nucleic acid. When nucleic acids of known sequence are hybridised, the hybrid length may be determined by aligning the sequences and identifying the conserved regions described herein. 1×SSC is 0.15M NaCl and 15 mM sodium citrate; the hybridisation solution and wash solutions may additionally include 5×Denhardt's reagent, 0.5-1.0% SDS, 100 μg/ml denatured, fragmented salmon sperm DNA, 0.5% sodium pyrophosphate.
For the purposes of defining the level of stringency, reference can be made to Sambrook et al. (2001) Molecular Cloning: a laboratory manual, 3rd Edition, Cold Spring Harbor Laboratory Press, CSH, New York or to Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989 and yearly updates).
Splice VariantThe term “splice variant” as used herein encompasses variants of a nucleic acid sequence in which selected introns and/or exons have been excised, replaced, displaced or added, or in which introns have been shortened or lengthened. Such variants will be ones in which the biological activity of the protein is substantially retained; this may be achieved by selectively retaining functional segments of the protein. Such splice variants may be found in nature or may be manmade. Methods for predicting and isolating such splice variants are well known in the art (see for example Foissac and Schiex (2005) BMC Bioinformatics 6: 25).
Allelic VariantAlleles or allelic variants are alternative forms of a given gene, located at the same chromosomal position. Allelic variants encompass Single Nucleotide Polymorphisms (SNPs), as well as Small Insertion/Deletion Polymorphisms (INDELs). The size of INDELs is usually less than 100 bp. SNPs and INDELs form the largest set of sequence variants in naturally occurring polymorphic strains of most organisms.
Gene Shuffling/Directed EvolutionGene shuffling or directed evolution consists of iterations of DNA shuffling followed by appropriate screening and/or selection to generate variants of nucleic acids or portions thereof encoding proteins having a modified biological activity (Castle et al., (2004) Science 304(5674): 1151-4; U.S. Pat. Nos. 5,811,238 and 6,395,547).
Regulatory Element/Control Sequence/PromoterThe terms “regulatory element”, “control sequence” and “promoter” are all used interchangeably herein and are to be taken in a broad context to refer to regulatory nucleic acid sequences capable of effecting expression of the sequences to which they are ligated. The term “promoter” typically refers to a nucleic acid control sequence located upstream from the transcriptional start of a gene and which is involved in recognising and binding of RNA polymerase and other proteins, thereby directing transcription of an operably linked nucleic acid. Encompassed by the aforementioned terms are transcriptional regulatory sequences derived from a classical eukaryotic genomic gene (including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence) and additional regulatory elements (i.e. upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or external stimuli, or in a tissue-specific manner. Also included within the term is a transcriptional regulatory sequence of a classical prokaryotic gene, in which case it may include a −35 box sequence and/or −10 box transcriptional regulatory sequences. The term “regulatory element” also encompasses a synthetic fusion molecule or derivative that confers, activates or enhances expression of a nucleic acid molecule in a cell, tissue or organ.
A “plant promoter” comprises regulatory elements, which mediate the expression of a coding sequence segment in plant cells. Accordingly, a plant promoter need not be of plant origin, but may originate from viruses or micro-organisms, for example from viruses which attack plant cells. The “plant promoter” can also originate from a plant cell, e.g. from the plant which is transformed with the nucleic acid sequence to be expressed in the inventive process and described herein. This also applies to other “plant” regulatory signals, such as “plant” terminators. The promoters upstream of the nucleotide sequences useful in the methods of the present invention can be modified by one or more nucleotide substitution(s), insertion(s) and/or deletion(s) without interfering with the functionality or activity of either the promoters, the open reading frame (ORF) or the 3′-regulatory region such as terminators or other 3′ regulatory regions which are located away from the ORF. It is furthermore possible that the activity of the promoters is increased by modification of their sequence, or that they are replaced completely by more active promoters, even promoters from heterologous organisms. For expression in plants, the nucleic acid molecule must, as described above, be linked operably to or comprise a suitable promoter which expresses the gene at the right point in time and with the required spatial expression pattern.
For the identification of functionally equivalent promoters, the promoter strength and/or expression pattern of a candidate promoter may be analysed for example by operably linking the promoter to a reporter gene and assaying the expression level and pattern of the reporter gene in various tissues of the plant. Suitable well-known reporter genes include for example beta-glucuronidase or beta-galactosidase. The promoter activity is assayed by measuring the enzymatic activity of the beta-glucuronidase or beta-galactosidase. The promoter strength and/or expression pattern may then be compared to that of a reference promoter (such as the one used in the methods of the present invention). Alternatively, promoter strength may be assayed by quantifying mRNA levels or by comparing mRNA levels of the nucleic acid used in the methods of the present invention, with mRNA levels of housekeeping genes such as 18S rRNA, using methods known in the art, such as Northern blotting with densitometric analysis of autoradiograms, quantitative real-time PCR or RT-PCR (Heid et al., 1996 Genome Methods 6: 986-994). Generally by “weak promoter” is intended a promoter that drives expression of a coding sequence at a low level. By “low level” is intended at levels of about 1/10,000 transcripts to about 1/100,000 transcripts, to about 1/500,0000 transcripts per cell. Conversely, a “strong promoter” drives expression of a coding sequence at high level, or at about 1/10 transcripts to about 1/100 transcripts to about 1/1000 transcripts per cell.
Generally, by “medium strength promoter” is intended a promoter that drives expression of a coding sequence at a lower level than a strong promoter, in particular at a level that is in all instances below that obtained when under the control of a 35S CaMV promoter. Typically a medium strength promoter is able to drive expression of a nucleic acid at least 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 times lower that of the 35S CaMV promoter.
Operably LinkedThe term “operably linked” as used herein refers to a functional linkage between the promoter sequence and the gene of interest, such that the promoter sequence is able to initiate transcription of the gene of interest.
Constitutive PromoterA “constitutive promoter” refers to a promoter that is transcriptionally active during most, but not necessarily all, phases of growth and development and under most environmental conditions, in at least one cell, tissue or organ. Table 2 below gives examples of constitutive promoters.
A ubiquitous promoter is active in substantially all tissues or cells of an organism.
Developmentally-Regulated PromoterA developmentally-regulated promoter is active during certain developmental stages or in parts of the plant that undergo developmental changes.
Inducible PromoterAn inducible promoter has induced or increased transcription initiation in response to a chemical (for a review see Gatz 1997, Annu. Rev. Plant Physiol. Plant Mol. Biol., 48:89-108), environmental or physical stimulus, or may be “stress-inducible”, i.e. activated when a plant is exposed to various stress conditions, or a “pathogen-inducible” i.e. activated when a plant is exposed to exposure to various pathogens.
Organ-Specific/Tissue-Specific PromoterAn organ-specific or tissue-specific promoter is one that is capable of preferentially initiating transcription in certain organs or tissues, such as the leaves, roots, seed tissue etc. For example, a “root-specific promoter” is a promoter that is transcriptionally active predominantly in plant roots, substantially to the exclusion of any other parts of a plant, whilst still allowing for any leaky expression in these other plant parts. Promoters able to initiate transcription in certain cells only are referred to herein as “cell-specific”.
Examples of root-specific promoters are listed in Table i below:
A seed-specific promoter is transcriptionally active predominantly in seed tissue, but not necessarily exclusively in seed tissue (in cases of leaky expression). The seed-specific promoter may be active during seed development and/or during germination. Examples of seed-specific promoters are given in Qing Qu and Takaiwa (Plant Biotechnol. J. 2, 113-125, 2004), which disclosure is incorporated by reference herein as if fully set forth and in Table ii below.
A green tissue-specific promoter as defined herein is a promoter that is transcriptionally active predominantly in green tissue, substantially to the exclusion of any other parts of a plant, whilst still allowing for any leaky expression in these other plant parts. Examples of green tissue-specific promoters which may be used to perform the methods of the invention are shown in Table iii below.
Another example of a tissue-specific promoter is a meristem-specific promoter, which is transcriptionally active predominantly in meristematic tissue, substantially to the exclusion of any other parts of a plant, whilst still allowing for any leaky expression in these other plant parts. Examples of meristem-specific promoters which may be used to perform the methods of the invention are shown in Table iv below.
The term “terminator” encompasses a control sequence which is a DNA sequence at the end of a transcriptional unit which signals 3′ processing and polyadenylation of a primary transcript and termination of transcription. The terminator can be derived from the natural gene, from a variety of other plant genes, or from T-DNA. The terminator to be added may be derived from, for example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or less preferably from any other eukaryotic gene.
ModulationThe term “modulation” means in relation to expression or gene expression, a process in which the expression level is changed by said gene expression in comparison to the control plant, the expression level may be increased or decreased. The original, unmodulated expression may be of any kind of expression of a structural RNA (rRNA, tRNA) or mRNA with subsequent translation. The term “modulating the activity” shall mean any change of the expression of the inventive nucleic acid sequences or encoded proteins, which leads to increased yield and/or increased growth of the plants.
Expression
The term “expression” or “gene expression” means the transcription of a specific gene or specific genes or specific genetic construct. The term “expression” or “gene expression” in particular means the transcription of a gene or genes or genetic construct into structural RNA (rRNA, tRNA) or mRNA with or without subsequent translation of the latter into a protein. The process includes transcription of DNA and processing of the resulting mRNA product.
Increased Expression/OverexpressionThe term “increased expression” or “overexpression” as used herein means any form of expression that is additional to the original wild-type expression level.
Methods for increasing expression of genes or gene products are well documented in the art and include, for example, overexpression driven by appropriate promoters, the use of transcription enhancers or translation enhancers. Isolated nucleic acids which serve as promoter or enhancer elements may be introduced in an appropriate position (typically upstream) of a non-heterologous form of a polynucleotide so as to upregulate expression of a nucleic acid encoding the polypeptide of interest. For example, endogenous promoters may be altered in vivo by mutation, deletion, and/or substitution (see, Kmiec, U.S. Pat. No. 5,565,350; Zarling et al., WO9322443), or isolated promoters may be introduced into a plant cell in the proper orientation and distance from a gene of the present invention so as to control the expression of the gene.
If polypeptide expression is desired, it is generally desirable to include a polyadenylation region at the 3′-end of a polynucleotide coding region. The polyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from T-DNA. The 3′ end sequence to be added may be derived from, for example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or less preferably from any other eukaryotic gene.
An intron sequence may also be added to the 5′ untranslated region (UTR) or the coding sequence of the partial coding sequence to increase the amount of the mature message that accumulates in the cytosol. Inclusion of a spliceable intron in the transcription unit in both plant and animal expression constructs has been shown to increase gene expression at both the mRNA and protein levels up to 1000-fold (Buchman and Berg (1988) Mol. Cell biol. 8: 4395-4405; Callis et al. (1987) Genes Dev 1:1183-1200). Such intron enhancement of gene expression is typically greatest when placed near the 5′ end of the transcription unit. Use of the maize introns Adh1-S intron 1, 2, and 6, the Bronze-1 intron are known in the art. For general information see: The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, N.Y. (1994).
Endogenous GeneReference herein to an “endogenous” gene not only refers to the gene in question as found in a plant in its natural form (i.e., without there being any human intervention), but also refers to that same gene (or a substantially homologous nucleic acid/gene) in an isolated form subsequently (re)introduced into a plant (a transgene). For example, a transgenic plant containing such a transgene may encounter a substantial reduction of the transgene expression and/or substantial reduction of expression of the endogenous gene. The isolated gene may be isolated from an organism or may be manmade, for example by chemical synthesis.
Decreased ExpressionReference herein to “decreased expression” or “reduction or substantial elimination” of expression is taken to mean a decrease in endogenous gene expression and/or polypeptide levels and/or polypeptide activity relative to control plants. The reduction or substantial elimination is in increasing order of preference at least 10%, 20%, 30%, 40% or 50%, 60%, 70%, 80%, 85%, 90%, or 95%, 96%, 97%, 98%, 99% or more reduced compared to that of control plants.
For the reduction or substantial elimination of expression an endogenous gene in a plant, a sufficient length of substantially contiguous nucleotides of a nucleic acid sequence is required. In order to perform gene silencing, this may be as little as 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10 or fewer nucleotides, alternatively this may be as much as the entire gene (including the 5′ and/or 3′ UTR, either in part or in whole). The stretch of substantially contiguous nucleotides may be derived from the nucleic acid encoding the Patellin, or the Precursor RNA Processing factor 38, or the Adaptor 2, or a WDR23-like polypeptide (target genes), or from any nucleic acid capable of encoding an orthologue, paralogue or homologue of the Patellin, or the Precursor RNA Processing factor 38, or the Adaptor 2, or a WDR23-like polypeptide. Preferably, the stretch of substantially contiguous nucleotides is capable of forming hydrogen bonds with the target gene (either sense or antisense strand), more preferably, the stretch of substantially contiguous nucleotides has, in increasing order of preference, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity to the target gene (either sense or antisense strand). A nucleic acid sequence encoding a (functional) polypeptide is not a requirement for the various methods discussed herein for the reduction or substantial elimination of expression of an endogenous gene.
Examples of various methods for the reduction or substantial elimination of expression in a plant of an endogenous gene, or for lowering levels and/or activity of a protein, are known to the skilled in the art. A skilled person would readily be able to adapt the known methods for silencing, so as to achieve reduction of expression of an endogenous gene in a whole plant or in parts thereof through the use of an appropriate promoter, for example.
This reduction or substantial elimination of expression may be achieved using routine tools and techniques. A preferred method for the reduction or substantial elimination of endogenous gene expression is by introducing and expressing in a plant a genetic construct into which the nucleic acid (in this case a stretch of substantially contiguous nucleotides derived from the gene of interest, or from any nucleic acid capable of encoding an orthologue, paralogue or homologue of any one of the Patellin, or the Precursor RNA Processing factor 38, or the Adaptor 2, or a WDR23-like polypeptide) is cloned as an inverted repeat (in part or completely), separated by a spacer (non-coding DNA).
Another method for the reduction or substantial elimination of endogenous gene expression is by RNA-mediated silencing using an inverted repeat of a nucleic acid sequence or a part thereof (in this case a stretch of substantially contiguous nucleotides derived from the gene of interest, or from any nucleic acid sequence capable of encoding an orthologue, paralogue or homologue of the Patellin, or the Precursor RNA Processing factor 38, or the Adaptor 2, or a WDR23-like polypeptide), preferably capable of forming a hairpin structure. Another example of an RNA silencing method involves the introduction of nucleic acid sequences or parts thereof (in this case a stretch of substantially contiguous nucleotides derived from the gene of interest, or from any nucleic acid sequence capable of encoding an orthologue, paralogue or homologue of the Patellin, or the Precursor RNA Processing factor 38, or the Adaptor 2, or a WDR23-like polypeptide) in a sense orientation into a plant. Another example of an RNA silencing method involves the use of antisense nucleic acid sequences. Gene silencing may also be achieved by insertion mutagenesis (for example, T-DNA insertion or transposon insertion) or by strategies as described by, among others, Angell and Baulcombe ((1999) Plant J 20(3): 357-62), (Amplicon VIGS WO 98/36083), or Baulcombe (WO 99/15682). Other methods, such as the use of antibodies directed to an endogenous polypeptide for inhibiting its function in planta, or interference in the signalling pathway in which a polypeptide is involved, will be well known to the skilled man. Artificial and/or natural microRNAs (miRNAs) may be used to knock out gene expression and/or mRNA translation. Endogenous miRNAs are single stranded small RNAs of typically 19-24 nucleotides long. Artificial microRNAs (amiRNAs), which are typically 21 nucleotides in length, can be genetically engineered specifically to negatively regulate gene expression of single or multiple genes of interest. Determinants of plant microRNA target selection are well known in the art. Empirical parameters for target recognition have been defined and can be used to aid in the design of specific amiRNAs (Schwab et al., (2005) Dev Cell 8(4):517-27). Convenient tools for design and generation of amiRNAs and their precursors are also available to the public (Schwab et al., (2006) Plant Cell 18(5):1121-33).
In such a preferred method, expression of the endogenous gene is reduced or substantially eliminated through RNA-mediated silencing using an inverted repeat of a nucleic acid or a part thereof (in this case a stretch of substantially contiguous nucleotides derived from the gene of interest, or from any nucleic acid capable of encoding an orthologue, paralogue or homologue of the Patellin, or the Precursor RNA Processing factor 38, or the Adaptor 2, or a WDR23-like polypeptide), preferably capable of forming a hairpin structure. The inverted repeat is cloned in an expression vector comprising control sequences. A non-coding DNA nucleic acid sequence (a spacer, for example a matrix attachment region fragment (MAR), an intron, a polylinker, etc.) is located between the two inverted nucleic acids forming the inverted repeat. After transcription of the inverted repeat, a chimeric RNA with a self-complementary structure is formed (partial or complete). This double-stranded RNA structure is referred to as the hairpin RNA (hpRNA). The hpRNA is processed by the plant into siRNAs that are incorporated into an RNA-induced silencing complex (RISC). The RISC further cleaves the mRNA transcripts, thereby substantially reducing the number of mRNA transcripts to be translated into polypeptides. For further general details see for example, Grierson et al. (1998) WO 98/53083; Waterhouse et al. (1999) WO 99/53050).
Performance of the methods of the invention does not rely on introducing and expressing in a plant a genetic construct into which the nucleic acid is cloned as an inverted repeat, but any one or more of several well-known “gene silencing” methods may be used to achieve the same effects.
One such method for the reduction of endogenous gene expression is RNA-mediated silencing of gene expression (downregulation). Silencing in this case is triggered in a plant by a double stranded RNA sequence (dsRNA) that is substantially similar to the target endogenous gene. This dsRNA is further processed by the plant into about 20 to about 26 nucleotides called short interfering RNAs (siRNAs). The siRNAs are incorporated into an RNA-induced silencing complex (RISC) that cleaves the mRNA transcript of the endogenous target gene, thereby substantially reducing the number of mRNA transcripts to be translated into a polypeptide. Preferably, the double stranded RNA sequence corresponds to a target gene.
Another example of an RNA silencing method involves the introduction of nucleic acid sequences or parts thereof (in this case a stretch of substantially contiguous nucleotides derived from the gene of interest, or from any nucleic acid capable of encoding an orthologue, paralogue or homologue of the Patellin, or the Precursor RNA Processing factor 38, or the Adaptor 2) in a sense orientation into a plant. “Sense orientation” refers to a DNA sequence that is homologous to an mRNA transcript thereof. Introduced into a plant would therefore be at least one copy of the nucleic acid sequence. The additional nucleic acid sequence will reduce expression of the endogenous gene, giving rise to a phenomenon known as co-suppression. The reduction of gene expression will be more pronounced if several additional copies of a nucleic acid sequence are introduced into the plant, as there is a positive correlation between high transcript levels and the triggering of co-suppression.
Another example of an RNA silencing method involves the use of antisense nucleic acid sequences. An “antisense” nucleic acid sequence comprises a nucleotide sequence that is complementary to a “sense” nucleic acid sequence encoding a protein, i.e. complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA transcript sequence. The antisense nucleic acid sequence is preferably complementary to the endogenous gene to be silenced. The complementarity may be located in the “coding region” and/or in the “non-coding region” of a gene. The term “coding region” refers to a region of the nucleotide sequence comprising codons that are translated into amino acid residues. The term “non-coding region” refers to 5′ and 3′ sequences that flank the coding region that are transcribed but not translated into amino acids (also referred to as 5′ and 3′ untranslated regions).
Antisense nucleic acid sequences can be designed according to the rules of Watson and Crick base pairing. The antisense nucleic acid sequence may be complementary to the entire nucleic acid sequence (in this case a stretch of substantially contiguous nucleotides derived from the gene of interest, or from any nucleic acid capable of encoding an orthologue, paralogue or homologue of the Patellin, or the Precursor RNA Processing factor 38, or the Adaptor 2), but may also be an oligonucleotide that is antisense to only a part of the nucleic acid sequence (including the mRNA 5′ and 3′ UTR). For example, the antisense oligonucleotide sequence may be complementary to the region surrounding the translation start site of an mRNA transcript encoding a polypeptide. The length of a suitable antisense oligonucleotide sequence is known in the art and may start from about 50, 45, 40, 35, 30, 25, 20, 15 or 10 nucleotides in length or less. An antisense nucleic acid sequence according to the invention may be constructed using chemical synthesis and enzymatic ligation reactions using methods known in the art. For example, an antisense nucleic acid sequence (e.g., an antisense oligonucleotide sequence) may be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acid sequences, e.g., phosphorothioate derivatives and acridine substituted nucleotides may be used. Examples of modified nucleotides that may be used to generate the antisense nucleic acid sequences are well known in the art. Known nucleotide modifications include methylation, cyclization and ‘caps’ and substitution of one or more of the naturally occurring nucleotides with an analogue such as inosine. Other modifications of nucleotides are well known in the art.
The antisense nucleic acid sequence can be produced biologically using an expression vector into which a nucleic acid sequence has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest). Preferably, production of antisense nucleic acid sequences in plants occurs by means of a stably integrated nucleic acid construct comprising a promoter, an operably linked antisense oligonucleotide, and a terminator.
The nucleic acid molecules used for silencing in the methods of the invention (whether introduced into a plant or generated in situ) hybridize with or bind to mRNA transcripts and/or genomic DNA encoding a polypeptide to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid sequence which binds to DNA duplexes, through specific interactions in the major groove of the double helix. Antisense nucleic acid sequences may be introduced into a plant by transformation or direct injection at a specific tissue site. Alternatively, antisense nucleic acid sequences can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense nucleic acid sequences can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid sequence to peptides or antibodies which bind to cell surface receptors or antigens. The antisense nucleic acid sequences can also be delivered to cells using the vectors described herein.
According to a further aspect, the antisense nucleic acid sequence is an a-anomeric nucleic acid sequence. An a-anomeric nucleic acid sequence forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual b-units, the strands run parallel to each other (Gaultier et al. (1987) Nucl Ac Res 15: 6625-6641). The antisense nucleic acid sequence may also comprise a 2′-o-methylribonucleotide (Inoue et al. (1987) Nucl Ac Res 15, 6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215, 327-330).
The reduction or substantial elimination of endogenous gene expression may also be performed using ribozymes. Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid sequence, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334, 585-591) can be used to catalytically cleave mRNA transcripts encoding a polypeptide, thereby substantially reducing the number of mRNA transcripts to be translated into a polypeptide. A ribozyme having specificity for a nucleic acid sequence can be designed (see for example: Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742). Alternatively, mRNA transcripts corresponding to a nucleic acid sequence can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules (Bartel and Szostak (1993) Science 261, 1411-1418). The use of ribozymes for gene silencing in plants is known in the art (e.g., Atkins et al. (1994) WO 94/00012; Lenne et al. (1995) WO 95/03404; Lutziger et al. (2000) WO 00/00619; Prinsen et al. (1997) WO 97/13865 and Scott et al. (1997) WO 97/38116).
Gene silencing may also be achieved by insertion mutagenesis (for example, T-DNA insertion or transposon insertion) or by strategies as described by, among others, Angell and Baulcombe ((1999) Plant J 20(3): 357-62), (Amplicon VIGS WO 98/36083), or Baulcombe (WO 99/15682).
Gene silencing may also occur if there is a mutation on an endogenous gene and/or a mutation on an isolated gene/nucleic acid subsequently introduced into a plant. The reduction or substantial elimination may be caused by a non-functional polypeptide. For example, the polypeptide may bind to various interacting proteins; one or more mutation(s) and/or truncation(s) may therefore provide for a polypeptide that is still able to bind interacting proteins (such as receptor proteins) but that cannot exhibit its normal function (such as signalling ligand).
A further approach to gene silencing is by targeting nucleic acid sequences complementary to the regulatory region of the gene (e.g., the promoter and/or enhancers) to form triple helical structures that prevent transcription of the gene in target cells. See Helene, C., Anticancer Drug Res. 6, 569-84, 1991; Helene et al., Ann. N.Y. Acad. Sci. 660, 27-36 1992; and Maher, L. J. Bioassays 14, 807-15, 1992.
Other methods, such as the use of antibodies directed to an endogenous polypeptide for inhibiting its function in planta, or interference in the signalling pathway in which a polypeptide is involved, will be well known to the skilled man. In particular, it can be envisaged that manmade molecules may be useful for inhibiting the biological function of a target polypeptide, or for interfering with the signalling pathway in which the target polypeptide is involved.
Alternatively, a screening program may be set up to identify in a plant population natural variants of a gene, which variants encode polypeptides with reduced activity. Such natural variants may also be used for example, to perform homologous recombination.
Artificial and/or natural microRNAs (miRNAs) may be used to knock out gene expression and/or mRNA translation. Endogenous miRNAs are single stranded small RNAs of typically 19-24 nucleotides long. They function primarily to regulate gene expression and/or mRNA translation. Most plant microRNAs (miRNAs) have perfect or near-perfect complementarity with their target sequences. However, there are natural targets with up to five mismatches. They are processed from longer non-coding RNAs with characteristic fold-back structures by double-strand specific RNases of the Dicer family. Upon processing, they are incorporated in the RNA-induced silencing complex (RISC) by binding to its main component, an Argonaute protein. MiRNAs serve as the specificity components of RISC, since they base-pair to target nucleic acids, mostly mRNAs, in the cytoplasm. Subsequent regulatory events include target mRNA cleavage and destruction and/or translational inhibition. Effects of miRNA overexpression are thus often reflected in decreased mRNA levels of target genes.
Artificial microRNAs (amiRNAs), which are typically 21 nucleotides in length, can be genetically engineered specifically to negatively regulate gene expression of single or multiple genes of interest. Determinants of plant microRNA target selection are well known in the art. Empirical parameters for target recognition have been defined and can be used to aid in the design of specific amiRNAs, (Schwab et al., Dev. Cell 8, 517-527, 2005). Convenient tools for design and generation of amiRNAs and their precursors are also available to the public (Schwab et al., Plant Cell 18, 1121-1133, 2006).
For optimal performance, the gene silencing techniques used for reducing expression in a plant of an endogenous gene requires the use of nucleic acid sequences from monocotyledonous plants for transformation of monocotyledonous plants, and from dicotyledonous plants for transformation of dicotyledonous plants. Preferably, a nucleic acid sequence from any given plant species is introduced into that same species. For example, a nucleic acid sequence from rice is transformed into a rice plant. However, it is not an absolute requirement that the nucleic acid sequence to be introduced originates from the same plant species as the plant in which it will be introduced. It is sufficient that there is substantial homology between the endogenous target gene and the nucleic acid to be introduced.
Described above are examples of various methods for the reduction or substantial elimination of expression in a plant of an endogenous gene. A person skilled in the art would readily be able to adapt the aforementioned methods for silencing so as to achieve reduction of expression of an endogenous gene in a whole plant or in parts thereof through the use of an appropriate promoter, for example.
Selectable Marker (Gene)/Reporter Gene“Selectable marker”, “selectable marker gene” or “reporter gene” includes any gene that confers a phenotype on a cell in which it is expressed to facilitate the identification and/or selection of cells that are transfected or transformed with a nucleic acid construct of the invention. These marker genes enable the identification of a successful transfer of the nucleic acid molecules via a series of different principles. Suitable markers may be selected from markers that confer antibiotic or herbicide resistance, that introduce a new metabolic trait or that allow visual selection. Examples of selectable marker genes include genes conferring resistance to antibiotics (such as nptII that phosphorylates neomycin and kanamycin, or hpt, phosphorylating hygromycin, or genes conferring resistance to, for example, bleomycin, streptomycin, tetracyclin, chloramphenicol, ampicillin, gentamycin, geneticin (G418), spectinomycin or blasticidin), to herbicides (for example bar which provides resistance to Basta®; aroA or gox providing resistance against glyphosate, or the genes conferring resistance to, for example, imidazolinone, phosphinothricin or sulfonylurea), or genes that provide a metabolic trait (such as manA that allows plants to use mannose as sole carbon source or xylose isomerase for the utilisation of xylose, or antinutritive markers such as the resistance to 2-deoxyglucose). Expression of visual marker genes results in the formation of colour (for example β-glucuronidase, GUS or β-galactosidase with its coloured substrates, for example X-Gal), luminescence (such as the luciferin/luceferase system) or fluorescence (Green Fluorescent Protein, GFP, and derivatives thereof). This list represents only a small number of possible markers. The skilled worker is familiar with such markers. Different markers are preferred, depending on the organism and the selection method.
It is known that upon stable or transient integration of nucleic acids into plant cells, only a minority of the cells takes up the foreign DNA and, if desired, integrates it into its genome, depending on the expression vector used and the transfection technique used. To identify and select these integrants, a gene coding for a selectable marker (such as the ones described above) is usually introduced into the host cells together with the gene of interest. These markers can for example be used in mutants in which these genes are not functional by, for example, deletion by conventional methods. Furthermore, nucleic acid molecules encoding a selectable marker can be introduced into a host cell on the same vector that comprises the sequence encoding the polypeptides of the invention or used in the methods of the invention, or else in a separate vector. Cells which have been stably transfected with the introduced nucleic acid can be identified for example by selection (for example, cells which have integrated the selectable marker survive whereas the other cells die). The marker genes may be removed or excised from the transgenic cell once they are no longer needed. Techniques for marker gene removal are known in the art, useful techniques are described above in the definitions section.
Since the marker genes, particularly genes for resistance to antibiotics and herbicides, are no longer required or are undesired in the transgenic host cell once the nucleic acids have been introduced successfully, the process according to the invention for introducing the nucleic acids advantageously employs techniques which enable the removal or excision of these marker genes. One such a method is what is known as co-transformation. The co-transformation method employs two vectors simultaneously for the transformation, one vector bearing the nucleic acid according to the invention and a second bearing the marker gene(s). A large proportion of transformants receives or, in the case of plants, comprises (up to 40% or more of the transformants), both vectors. In case of transformation with Agrobacteria, the transformants usually receive only a part of the vector, i.e. the sequence flanked by the T-DNA, which usually represents the expression cassette. The marker genes can subsequently be removed from the transformed plant by performing crosses. In another method, marker genes integrated into a transposon are used for the transformation together with desired nucleic acid (known as the Ac/Ds technology). The transformants can be crossed with a transposase source or the transformants are transformed with a nucleic acid construct conferring expression of a transposase, transiently or stable. In some cases (approx. 10%), the transposon jumps out of the genome of the host cell once transformation has taken place successfully and is lost. In a further number of cases, the transposon jumps to a different location. In these cases the marker gene must be eliminated by performing crosses. In microbiology, techniques were developed which make possible, or facilitate, the detection of such events. A further advantageous method relies on what is known as recombination systems; whose advantage is that elimination by crossing can be dispensed with. The best-known system of this type is what is known as the Cre/lox system. Cre1 is a recombinase that removes the sequences located between the loxP sequences. If the marker gene is integrated between the loxP sequences, it is removed once transformation has taken place successfully, by expression of the recombinase. Further recombination systems are the HIN/HIX, FLP/FRT and REP/STB system (Tribble et al., J. Biol. Chem., 275, 2000: 22255-22267; Velmurugan et al., J. Cell Biol., 149, 2000: 553-566). A site-specific integration into the plant genome of the nucleic acid sequences according to the invention is possible. Naturally, these methods can also be applied to microorganisms such as yeast, fungi or bacteria.
Transgenic/Transgene/RecombinantFor the purposes of the invention, “transgenic”, “transgene” or “recombinant” means with regard to, for example, a nucleic acid sequence, an expression cassette, gene construct or a vector comprising the nucleic acid sequence or an organism transformed with the nucleic acid sequences, expression cassettes or vectors according to the invention, all those constructions brought about by recombinant methods in which either
-
- (a) the nucleic acid sequences encoding proteins useful in the methods of the invention, or
- (b) genetic control sequence(s) which is operably linked with the nucleic acid sequence according to the invention, for example a promoter, or
- (c) a) and b)
are not located in their natural genetic environment or have been modified by recombinant methods, it being possible for the modification to take the form of, for example, a substitution, addition, deletion, inversion or insertion of one or more nucleotide residues. The natural genetic environment is understood as meaning the natural genomic or chromosomal locus in the original plant or the presence in a genomic library. In the case of a genomic library, the natural genetic environment of the nucleic acid sequence is preferably retained, at least in part. The environment flanks the nucleic acid sequence at least on one side and has a sequence length of at least 50 bp, preferably at least 500 bp, especially preferably at least 1000 bp, most preferably at least 5000 bp. A naturally occurring expression cassette—for example the naturally occurring combination of the natural promoter of the nucleic acid sequences with the corresponding nucleic acid sequence encoding a polypeptide useful in the methods of the present invention, as defined above—becomes a transgenic expression cassette when this expression cassette is modified by non-natural, synthetic (“artificial”) methods such as, for example, mutagenic treatment. Suitable methods are described, for example, in U.S. Pat. No. 5,565,350 or WO 00/15815.
A transgenic plant for the purposes of the invention is thus understood as meaning, as above, that the nucleic acids used in the method of the invention are not at their natural locus in the genome of said plant, it being possible for the nucleic acids to be expressed homologously or heterologously. However, as mentioned, transgenic also means that, while the nucleic acids according to the invention or used in the inventive method are at their natural position in the genome of a plant, the sequence has been modified with regard to the natural sequence, and/or that the regulatory sequences of the natural sequences have been modified.
Transgenic is preferably understood as meaning the expression of the nucleic acids according to the invention at an unnatural locus in the genome, i.e. homologous or, preferably, heterologous expression of the nucleic acids takes place. Preferred transgenic plants are mentioned herein.
TransformationThe term “introduction” or “transformation” as referred to herein encompasses the transfer of an exogenous polynucleotide into a host cell, irrespective of the method used for transfer. Plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a genetic construct of the present invention and a whole plant regenerated there from. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed. Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem). The polynucleotide may be transiently or stably introduced into a host cell and may be maintained non-integrated, for example, as a plasmid. Alternatively, it may be integrated into the host genome. The resulting transformed plant cell may then be used to regenerate a transformed plant in a manner known to persons skilled in the art.
The transfer of foreign genes into the genome of a plant is called transformation. Transformation of plant species is now a fairly routine technique. Advantageously, any of several transformation methods may be used to introduce the gene of interest into a suitable ancestor cell. The methods described for the transformation and regeneration of plants from plant tissues or plant cells may be utilized for transient or for stable transformation. Transformation methods include the use of liposomes, electroporation, chemicals that increase free DNA uptake, injection of the DNA directly into the plant, particle gun bombardment, transformation using viruses or pollen and microprojection. Methods may be selected from the calcium/polyethylene glycol method for protoplasts (Krens, F. A. et al., (1982) Nature 296, 72-74; Negrutiu I et al. (1987) Plant Mol Biol 8: 363-373); electroporation of protoplasts (Shillito R. D. et al. (1985) Bio/Technol 3, 1099-1102); microinjection into plant material (Crossway A et al., (1986) Mol. Gen Genet 202: 179-185); DNA or RNA-coated particle bombardment (Klein T M et al., (1987) Nature 327: 70) infection with (non-integrative) viruses and the like. Transgenic plants, including transgenic crop plants, are preferably produced via Agrobacterium-mediated transformation. An advantageous transformation method is the transformation in planta. To this end, it is possible, for example, to allow the agrobacteria to act on plant seeds or to inoculate the plant meristem with agrobacteria. It has proved particularly expedient in accordance with the invention to allow a suspension of transformed agrobacteria to act on the intact plant or at least on the flower primordia. The plant is subsequently grown on until the seeds of the treated plant are obtained (Clough and Bent, Plant J. (1998) 16, 735-743). Methods for Agrobacterium-mediated transformation of rice include well known methods for rice transformation, such as those described in any of the following: European patent application EP 1198985 A1, Aldemita and Hodges (Planta 199: 612-617, 1996); Chan et al. (Plant Mol Biol 22 (3): 491-506, 1993), Hiei et al. (Plant J 6 (2): 271-282, 1994), which disclosures are incorporated by reference herein as if fully set forth. In the case of corn transformation, the preferred method is as described in either Ishida et al. (Nat. Biotechnol 14(6): 745-50, 1996) or Frame et al. (Plant Physiol 129(1): 13-22, 2002), which disclosures are incorporated by reference herein as if fully set forth. Said methods are further described by way of example in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, eds. S. D. Kung and R. Wu, Academic Press (1993) 128-143 and in Potrykus Annu. Rev. Plant Physiol. Plant Molec. Biol. 42 (1991) 205-225). The nucleic acids or the construct to be expressed is preferably cloned into a vector, which is suitable for transforming Agrobacterium tumefaciens, for example pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984) 8711). Agrobacteria transformed by such a vector can then be used in known manner for the transformation of plants, such as plants used as a model, like Arabidopsis (Arabidopsis thaliana is within the scope of the present invention not considered as a crop plant), or crop plants such as, by way of example, tobacco plants, for example by immersing bruised leaves or chopped leaves in an agrobacterial solution and then culturing them in suitable media. The transformation of plants by means of Agrobacterium tumefaciens is described, for example, by Hofgen and Willmitzer in Nucl. Acid Res. (1988) 16, 9877 or is known inter alia from F. F. White, Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, Vol. 1, Engineering and Utilization, eds. S. D. Kung and R. Wu, Academic Press, 1993, pp. 15-38.
In addition to the transformation of somatic cells, which then have to be regenerated into intact plants, it is also possible to transform the cells of plant meristems and in particular those cells which develop into gametes. In this case, the transformed gametes follow the natural plant development, giving rise to transgenic plants. Thus, for example, seeds of Arabidopsis are treated with agrobacteria and seeds are obtained from the developing plants of which a certain proportion is transformed and thus transgenic [Feldman, K A and Marks M D (1987). Mol Gen Genet 208:274-289; Feldmann K (1992). In: C Koncz, N-H Chua and J Shell, eds, Methods in Arabidopsis Research. Word Scientific, Singapore, pp. 274-289]. Alternative methods are based on the repeated removal of the inflorescences and incubation of the excision site in the center of the rosette with transformed agrobacteria, whereby transformed seeds can likewise be obtained at a later point in time (Chang (1994). Plant J. 5: 551-558; Katavic (1994). Mol Gen Genet, 245: 363-370). However, an especially effective method is the vacuum infiltration method with its modifications such as the “floral dip” method. In the case of vacuum infiltration of Arabidopsis, intact plants under reduced pressure are treated with an agrobacterial suspension [Bechthold, N (1993). C R Acad Sci Paris Life Sci, 316: 1194-1199], while in the case of the “floral dip” method the developing floral tissue is incubated briefly with a surfactant-treated agrobacterial suspension [Clough, S J and Bent A F (1998) The Plant J. 16, 735-743]. A certain proportion of transgenic seeds are harvested in both cases, and these seeds can be distinguished from non-transgenic seeds by growing under the above-described selective conditions. In addition the stable transformation of plastids is of advantages because plastids are inherited maternally is most crops reducing or eliminating the risk of transgene flow through pollen. The transformation of the chloroplast genome is generally achieved by a process which has been schematically displayed in Klaus et al., 2004 [Nature Biotechnology 22 (2), 225-229]. Briefly the sequences to be transformed are cloned together with a selectable marker gene between flanking sequences homologous to the chloroplast genome. These homologous flanking sequences direct site specific integration into the plastome. Plastidal transformation has been described for many different plant species and an overview is given in Bock (2001) Transgenic plastids in basic research and plant biotechnology. J Mol Biol. 2001 Sep. 21; 312 (3):425-38 or Maliga, P (2003) Progress towards commercialization of plastid transformation technology. Trends Biotechnol. 21, 20-28. Further biotechnological progress has recently been reported in form of marker free plastid transformants, which can be produced by a transient co-integrated maker gene (Klaus et al., 2004, Nature Biotechnology 22(2), 225-229).
T-DNA Activation TaggingT-DNA activation tagging (Hayashi et al. Science (1992) 1350-1353), involves insertion of T-DNA, usually containing a promoter (may also be a translation enhancer or an intron), in the genomic region of the gene of interest or 10 kb up- or downstream of the coding region of a gene in a configuration such that the promoter directs expression of the targeted gene. Typically, regulation of expression of the targeted gene by its natural promoter is disrupted and the gene falls under the control of the newly introduced promoter. The promoter is typically embedded in a T-DNA. This T-DNA is randomly inserted into the plant genome, for example, through Agrobacterium infection and leads to modified expression of genes near the inserted T-DNA. The resulting transgenic plants show dominant phenotypes due to modified expression of genes close to the introduced promoter.
TILLINGThe term “TILLING” is an abbreviation of “Targeted Induced Local Lesions In Genomes” and refers to a mutagenesis technology useful to generate and/or identify nucleic acids encoding proteins with modified expression and/or activity. TILLING also allows selection of plants carrying such mutant variants. These mutant variants may exhibit modified expression, either in strength or in location or in timing (if the mutations affect the promoter for example). These mutant variants may exhibit higher activity than that exhibited by the gene in its natural form. TILLING combines high-density mutagenesis with high-throughput screening methods. The steps typically followed in TILLING are: (a) EMS mutagenesis (Redei G P and Koncz C (1992) In Methods in Arabidopsis Research, Koncz C, Chua N H, Schell J, eds. Singapore, World Scientific Publishing Co, pp. 16-82; Feldmann et al., (1994) In Meyerowitz E M, Somerville C R, eds, Arabidopsis. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp 137-172; Lightner J and Caspar T (1998) In J Martinez-Zapater, J Salinas, eds, Methods on Molecular Biology, Vol. 82. Humana Press, Totowa, N.J., pp 91-104); (b) DNA preparation and pooling of individuals; (c) PCR amplification of a region of interest; (d) denaturation and annealing to allow formation of heteroduplexes; (e) DHPLC, where the presence of a heteroduplex in a pool is detected as an extra peak in the chromatogram; (f) identification of the mutant individual; and (g) sequencing of the mutant PCR product. Methods for TILLING are well known in the art (McCallum et al., (2000) Nat Biotechnol 18: 455-457; reviewed by Stemple (2004) Nat Rev Genet 5(2): 145-50).
Homologous RecombinationHomologous recombination allows introduction in a genome of a selected nucleic acid at a defined selected position. Homologous recombination is a standard technology used routinely in biological sciences for lower organisms such as yeast or the moss Physcomitrella. Methods for performing homologous recombination in plants have been described not only for model plants (Offringa et al. (1990) EMBO J 9(10): 3077-84) but also for crop plants, for example rice (Terada et al. (2002) Nat Biotech 20(10): 1030-4; Iida and Terada (2004) Curr Opin Biotech 15(2): 132-8), and approaches exist that are generally applicable regardless of the target organism (Miller et al, Nature Biotechnol. 25, 778-785, 2007).
YieldThe term “yield” in general means a measurable produce of economic value, typically related to a specified crop, to an area, and to a period of time. Individual plant parts directly contribute to yield based on their number, size and/or weight, or the actual yield is the yield per square meter for a crop and year, which is determined by dividing total production (includes both harvested and appraised production) by planted square meters. The term “yield” of a plant may relate to vegetative biomass (root and/or shoot biomass), to reproductive organs, and/or to propagules (such as seeds) of that plant.
Early Vigour“Early vigour” refers to active healthy well-balanced growth especially during early stages of plant growth, and may result from increased plant fitness due to, for example, the plants being better adapted to their environment (i.e. optimizing the use of energy resources and partitioning between shoot and root). Plants having early vigour also show increased seedling survival and a better establishment of the crop, which often results in highly uniform fields (with the crop growing in uniform manner, i.e. with the majority of plants reaching the various stages of development at substantially the same time), and often better and higher yield. Therefore, early vigour may be determined by measuring various factors, such as thousand kernel weight, percentage germination, percentage emergence, seedling growth, seedling height, root length, root and shoot biomass and many more.
Increase/Improve/EnhanceThe terms “increase”, “improve” or “enhance” are interchangeable and shall mean in the sense of the application at least a 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%, preferably at least 15% or 20%, more preferably 25%, 30%, 35% or 40% more yield and/or growth in comparison to control plants as defined herein.
Seed YieldIncreased seed yield may manifest itself as one or more of the following: a) an increase in seed biomass (total seed weight) which may be on an individual seed basis and/or per plant and/or per square meter; b) increased number of flowers per plant; c) increased number of (filled) seeds; d) increased seed filling rate (which is expressed as the ratio between the number of filled seeds divided by the total number of seeds); e) increased harvest index, which is expressed as a ratio of the yield of harvestable parts, such as seeds, divided by the total biomass; f) increased thousand kernel weight (TKW) and g) increased number of primary panicles, which is extrapolated from the number of filled seeds counted and their total weight. An increased TKW may result from an increased seed size and/or seed weight, and may also result from an increase in embryo and/or endosperm size.
An increase in seed yield may also be manifested as an increase in seed size and/or seed volume. Furthermore, an increase in seed yield may also manifest itself as an increase in seed area and/or seed length and/or seed width and/or seed perimeter. Increased seed yield may also result in modified architecture, or may occur because of modified architecture.
Greenness IndexThe “greenness index” as used herein is calculated from digital images of plants. For each pixel belonging to the plant object on the image, the ratio of the green value versus the red value (in the RGB model for encoding color) is calculated. The greenness index is expressed as the percentage of pixels for which the green-to-red ratio exceeds a given threshold. Under normal growth conditions, under salt stress growth conditions, and under reduced nutrient availability growth conditions, the greenness index of plants is measured in the last imaging before flowering. In contrast, under drought stress growth conditions, the greenness index of plants is measured in the first imaging after drought.
PlantThe term “plant” as used herein encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, shoots, stems, leaves, roots (including tubers), flowers, and tissues and organs, wherein each of the aforementioned comprise the gene/nucleic acid of interest. The term “plant” also encompasses plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores, again wherein each of the aforementioned comprises the gene/nucleic acid of interest.
Plants that are particularly useful in the methods of the invention include all plants which belong to the superfamily Viridiplantae, in particular monocotyledonous and dicotyledonous plants including fodder or forage legumes, ornamental plants, food crops, trees or shrubs selected from the list comprising Acer spp., Actinidia spp., Abelmoschus spp., Agave sisalana, Agropyron spp., Agrostis stolonifera, Allium spp., Amaranthus spp., Ammophila arenaria, Ananas comosus, Annona spp., Apium graveolens, Arachis spp, Artocarpus spp., Asparagus officinalis, Avena spp. (e.g. Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida), Averrhoa carambola, Bambusa sp., Benincasa hispida, Bertholletia excelsea, Beta vulgaris, Brassica spp. (e.g. Brassica napus, Brassica rapa ssp. [canola, oilseed rape, turnip rape]), Cadaba farinosa, Camellia sinensis, Canna indica, Cannabis sativa, Capsicum spp., Carex elata, Carica papaya, Carissa macrocarpa, Carya spp., Carthamus tinctorius, Castanea spp., Ceiba pentandra, Cichorium endivia, Cinnamomum spp., Citrullus lanatus, Citrus spp., Cocos spp., Coffea spp., Colocasia esculenta, Cola spp., Corchorus sp., Coriandrum sativum, Corylus spp., Crataegus spp., Crocus sativus, Cucurbita spp., Cucumis spp., Cynara spp., Daucus carota, Desmodium spp., Dimocarpus longan, Dioscorea spp., Diospyros spp., Echinochloa spp., Elaeis (e.g. Elaeis guineensis, Elaeis oleifera), Eleusine coracana, Erianthus sp., Eriobotrya japonica, Eucalyptus sp., Eugenia uniflora, Fagopyrum spp., Fagus spp., Festuca arundinacea, Ficus carica, Fortunella spp., Fragaria spp., Ginkgo biloba, Glycine spp. (e.g. Glycine max, Soja hispida or Soja max), Gossypium hirsutum, Helianthus spp. (e.g. Helianthus annuus), Hemerocallis fulva, Hibiscus spp., Hordeum spp. (e.g. Hordeum vulgare), Ipomoea batatas, Juglans spp., Lactuca sativa, Lathyrus spp., Lens culinaris, Linum usitatissimum, Litchi chinensis, Lotus spp., Luffa acutangula, Lupinus spp., Luzula sylvatica, Lycopersicon spp. (e.g. Lycopersicon esculentum, Lycopersicon lycopersicum, Lycopersicon pyriforme), Macrotyloma spp., Malus spp., Malpighia emarginata, Mammea americana, Mangifera indica, Manihot spp., Manilkara zapota, Medicago sativa, Melilotus spp., Mentha spp., Miscanthus sinensis, Momordica spp., Morus nigra, Musa spp., Nicotiana spp., Olea spp., Opuntia spp., Ornithopus spp., Oryza spp. (e.g. Oryza sativa, Oryza latifolia), Panicum miliaceum, Panicum virgatum, Passiflora edulis, Pastinaca sativa, Pennisetum sp., Persea spp., Petroselinum crispum, Phalaris arundinacea, Phaseolus spp., Phleum pratense, Phoenix spp., Phragmites australis, Physalis spp., Pinus spp., Pistacia vera, Pisum spp., Poa spp., Populus spp., Prosopis spp., Prunus spp., Psidium spp., Punica granatum, Pyrus communis, Quercus spp., Raphanus sativus, Rheum rhabarbarum, Ribes spp., Ricinus communis, Rubus spp., Saccharum spp., Salix sp., Sambucus spp., Secale cereale, Sesamum spp., Sinapis sp., Solanum spp. (e.g. Solanum tuberosum, Solanum integrifolium or Solanum lycopersicum), Sorghum bicolor, Spinacia spp., Syzygium spp., Tagetes spp., Tamarindus indica, Theobroma cacao, Trifolium spp., Triticale sp., Triticosecale rimpaui, Triticum spp. (e.g. Triticum aestivum, Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha, Triticum sativum or Triticum vulgare), Tropaeolum minus, Tropaeolum majus, Vaccinium spp., Vicia spp., Vigna spp., Viola odorata, Vitis spp., Zea mays, Zizania palustris, Ziziphus spp., amongst others.
DETAILED DESCRIPTION OF THE INVENTIONSurprisingly, it has now been found that modulating expression in a plant of a nucleic acid encoding a PATL polypeptide, or a PRP38 polypeptide, or an GATA-like polypeptide, or an ADA2 polypeptide, or a WDR23-like polypeptide gives plants having enhanced yield-related traits relative to control plants. According to a first embodiment, the present invention provides a method for enhancing yield-related traits in plants relative to control plants, comprising modulating expression in a plant of a nucleic acid encoding a PATL polypeptide, or a PRP38 polypeptide, or an GATA-like polypeptide, or an ADA2 polypeptide, or a WDR23-like polypeptide and optionally selecting plants having enhanced yield-related traits.
In one embodiment, surprisingly, it has now been found that modulating (preferably increasing) expression in a plant of a nucleic acid encoding a GATA-like polypeptide gives plants having increased Thousand Kernel Weight (TKW) relative to control plants. According to a one embodiment, the present invention provides a method for enhancing TKW in plants relative to control plants, comprising modulating (preferably increasing) expression in a plant of a nucleic acid encoding a GATA-like polypeptide.
In another embodiment, it has now been found that increasing expression in a plant of a nucleic acid sequence encoding a WDR23-like polypeptide as defined herein, gives plants having increased yield-related traits relative to control plants. The invention provides nucleic acid sequences encoding WDR23-like polypeptides and WDR23-like polypeptides, whereby increased expression of the isolated nucleic acid sequences in plants increase yield-related traits relative to control plants.
A preferred method for modulating (preferably, increasing) expression of a nucleic acid encoding a PATL polypeptide, or a PRP38 polypeptide, or a GATA-like polypeptide, or ADA2 polypeptide, or a WDR23-like polypeptide is by introducing and expressing in a plant a nucleic acid encoding a PATL polypeptide, or a PRP38 polypeptide, or GATA-like polypeptide, or ADA2 polypeptide, or a WDR23-like polypeptide and optionally selecting for plants having enhanced yield-related traits.
Any reference hereinafter to a “protein useful in the methods of the invention” is taken to mean a PATL polypeptide, or a PRP38 polypeptide, or GATA-like polypeptide, or ADA2 polypeptide, or WDR23-like polypeptide as defined herein. Any reference hereinafter to a “nucleic acid useful in the methods of the invention” is taken to mean a nucleic acid capable of encoding such a PATL polypeptide, or a PRP38 polypeptide, or GATA-like polypeptide, or ADA2 polypeptide, or WDR23-like polypeptide. The nucleic acid to be introduced into a plant (and therefore useful in performing the methods of the invention) is any nucleic acid encoding the type of protein which will now be described, also named “PATL nucleic acid” or “PATL gene” or PRP38 nucleic acid” or “PRP38 gene”, or “GATA-like nucleic acid” or “GATA-like gene”, or “ADA2 nucleic acid” or “ADA2 gene”, or “WDR23-like nucleic acid sequence” or “WDR23-like gene”.
A “PATL polypeptide” as defined herein refers to any polypeptide comprising:
-
- (i) a SEC14 domain and/or
- (ii) a GOLD domain
The SEC 14 and GOLD domains are typically located in the C-terminal region of PATL polypeptides. The N-terminus of PATL polypeptides is variable in length and has a more divergent amino acid sequence composition than the C-terminus. The N-terminus is characterized by enrichment in acidic amino acids (PI— isoelectric poing—approximately 4) such as Glutamic acid (E) and contains numerous EEK (Glutamic-Glutamic-Lysine) repeats. In addition the N-terminus sequence is predicted to comprise one or more coiled coil, which are a common protein oligomerization-folding motif. Coiled coil domain usually contain hydrophobic and hydrophilic amino acid residues that form alpha-helices which in a cytoplasmic enviroment may wrap the hydrophobic strands against each other between the hydrophilic amino acids providing thermodynamic driving force for dimerization. Coiled coils may be readily identified using methods and software well described in the art such as COILs or PAIRCIOL2 (Lupas, et al; 1991. Science, 252, 1162-1164; MacDonnell et al. 2006. Bioinformatics; 22(3):356-8.
Preferred PATL polypeptides useful in the methods of the invention comprise an acidic N-terminus having one or more of the following features:
-
- (i) an isoelectric point in increasing order of preference of below 5, 4.5, 4, 3.5, or 3;
- (ii) one or more coiled coils, preferably in increasing order of preference one, two, three, four or five.
- (iii) one or more EEK repeats, preferably in increasing order of preference one, two, three, four, five, six or seven.
The N-terminus of a PATL polypeptide as referred herein is the portion of the protein extending at the N-terminus of the SEC14 domain.
The isoelectric point of a polypeptide, that is, the pH at which a particular molecule or surface carries no net electrical charge, may be calculated by methods well known in the art (Stryer, 1995) using for example the Henderson-Hasselbalch equation (Henderson (1908), Am. J. Physiol., 21, 173-179; Hasselbalc (1917), Biochemische Zeitschrift, 78, 112-144. de Livie, (2003) J. Chem. Educ., 80, 146).
Typically, PATL polypeptides comprise a SEC14 domain following the above-described N-terminus. The SEC14 domain in PATL polypeptides includes a region of homology to the yeast Sec14p protein which comprises the phospholipid binding pocket. The E, K and G amino acid residues involved in lipid binding and/or transfer in the yeast Sec14p are conserved in PATL polypeptides, which in SEQ ID NO: 2 correspond to E434, K465, and G493 (
PATL polypeptides may comprise a GOLD domain which is typically found at the C-terminus. The GOLD domain in PATL polypeptide is rich in Lysine residues and contains the conserved sequence KX(10-11)(K/R/T)KKKX(0-1)(L/V/A)(L/V/A)YR (SEQ ID NO: 286; see
A preferred PATL polypeptide useful in the methods of the invention comprises at least one of the following domains:
- (i) A SEC14 domain as represented by SEQ ID NO: 71: lpeldsvvfyrgadreghpvcynyygefqdkdlyekafgdeekrerflkwriqllergilsqldfspsgicsmvqvtdlknsp pmlgkhravtrgavallqdnypefiakkvfinvpwwylaankmmspfltqrtkskfifaspaksaetlfryiapegypvqfg glfk, or a domain having in increasing order of preference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82%, 85%, 90%, 92%, 95%, 97% or more sequence identity to the domain represented by SEQ ID NO: 71, or to any SEC14 domain as present in any of the polypeptides of Table A;
- (ii) A GOLD domain as represented by SEQ ID NO: 72: sdayteltikpssketveipvtenstigwelrylgwevsygaeftpdaeggytvivqktrkvpaneepimkgsfkvgepgki vltinnpaskkkkllyrskv, or a domain having in increasing order of preference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82%, 85%, 90%, 92%, 95%, 97% or more sequence identity to the domain represented by SEQ ID NO: 72, or to any GOLD domain as present in any of the polypeptides of Table A.
Further preferably a PATL polypeptide useful in the methods of the invention comprise at least one of the conserved motifs Motif Ia and Motif IIa as represented by SEQ ID NO: 69/L(L/T)KFLRAR and SEQ ID NO: 70/(L/F)(Q/E)DNYPEF respectively.
The PATL polypeptides useful in the methods of the invention are preferably those having in increasing order of preference at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 92%, 93%, 95%, 96%, 98% or more sequence identity to any of polypeptide given in Table A.
A SEC14 domain and a GOLD domain comprised in a PATL polypeptide may be identified by searching specialized databases containing collections of multiple sequence alignments and hidden Markov models covering conserved protein domains and families, such as Pfam which available at the Sanger Institute, United Kingdom. Alternatively the domains abovementioned may be found by scanning The Integrated Resource of Protein Families, Domains and Sites (InterPro) database to detect a significant sequence alignment with known SEC14 or GOLD domains (see Example 4). Interpro database is an integrated interface for the commonly used signature databases for text- and sequence-based searches. The InterPro database combines these databases, which use different methodologies and varying degrees of biological information about well-characterized proteins to derive protein signatures. Collaborating databases include SWISS-PROT, PROSITE, TrEMBL, PRINTS, ProDom and Pfam, Smart and TIGRFAMs. Interpro is hosted at the European Bioinformatics Institute in the United Kingdom. A significant alignment between the sequence of two polypeptides or two domains as defined herein is an alignment having in increasing order of preference an e-value lower than e−5 (e to the minus 5), 1.e−10, 1.e−15, 1.e−20, 1.e−25, 1.e−50, 1.e−100, 1.e−200, 1.e−300, 1.e−400, 1.e−500, 1.e−600, 1.e−700 and 1.e−800. The polypeptide sequences may be aligned using any of the methods well known in the art, including global and local alignment methods such as Blast algorithms, e.g. the algorithm described in Altschul, S F, et al. (1990) J. Mol. Biol. 215:403-10.
The probability for the alignment to occur with a given sequence is taken as basis for identifying similar polypeptides. A parameter that is typically used to represent such probability is called an e-value (E-value). The E-value is a measure of the reliability of the S score. The S score is a measure of the similarity of the two sequenced being aligned. The e-value describes how often a given S score is expected to occur at random. The e-value may be as high as 1.0.
Preferably PATL polypeptides useful in the methods of the invention comprise at least one of the following domains:
(i) a SEC14 domain having in increasing order of preference an e-value lower than e−5 (e to the minus 5), 1.e−10, 1.e−15, 1.e−20, 1.e−25, 1.e−50, 1.e−100, 1.e−200, 1.e−300, 1.e−400, 1.e−500, 1.e−600, 1.e−700 and 1.e−800 when aligned with a known SEC14 domain, more preferably when aligned to any of the SEC14 domains of Table C;
(ii) a GOLD domain having in increasing order of preference an e-value lower than e−5 (e to the minus 5), 1.e−10, 1.e−15, 1.e−20, 1.e−25, 1.e−50, 1.e−75, 1.e−100, 1.e−200, 1.e−300, 1.e−400, 1.e−500, 1.e−600, 1. e−700 and 1.e−800 when aligned with a known GOLD domain, more preferably when aligned to any of the GOLD domains of Table C.
Preferably, the PATL polypeptide sequence useful in the methods of the invention is one which when used in the construction of a phylogenetic tree, such as the one depicted in
The term “domain” and “motif” is defined in the “definitions” section herein. Specialist databases exist for the identification of domains, for example, SMART (Schultz et al. (1998) Proc. Natl. Acad. Sci. USA 95, 5857-5864; Letunic et al. (2002) Nucleic Acids Res 30, 242-244), InterPro (Mulder et al., (2003) Nucl. Acids. Res. 31, 315-318), Prosite (Bucher and Bairoch (1994), A generalized profile syntax for biomolecular sequences motifs and its function in automatic sequence interpretation. (In) ISMB-94; Proceedings 2nd International Conference on Intelligent Systems for Molecular Biology. Altman R., Brutlag D., Karp P., Lathrop R., Searls D., Eds., pp 53-61, AAAI Press, Menlo Park; Hulo et al., Nucl. Acids. Res. 32:D134-D137, (2004)), or Pfam (Bateman et al., Nucleic Acids Research 30(1): 276-280 (2002)). A set of tools for in silico analysis of protein sequences is available on the ExPASy proteomics server (Swiss Institute of Bioinformatics (Gasteiger et al., ExPASy: the proteomics server for in-depth protein knowledge and analysis, Nucleic Acids Res. 31:3784-3788(2003)). Domains or motifs may also be identified using routine techniques, such as by sequence alignment.
Methods for the alignment of sequences for comparison are well known in the art, such methods include GAP, BESTFIT, BLAST, FASTA and TFASTA. GAP uses the algorithm of Needleman and Wunsch ((1970) J Mol Biol 48: 443-453) to find the global (i.e. spanning the complete sequence) alignment of two sequences that maximizes the number of matches and minimizes the number of gaps. The BLAST algorithm (Altschul et al. (1990) J Mol Biol 215: 403-10) calculates percent sequence identity and performs a statistical analysis of the similarity between the two sequences. The software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information (NCBI). Homologues may readily be identified using, for example, the ClustalW multiple sequence alignment algorithm (version 1.83), with the default pairwise alignment parameters, and a scoring method in percentage. Global percentages of similarity and identity may also be determined using one of the methods available in the MatGAT software package (Campanella et al., BMC Bioinformatics. 2003 Jul. 10; 4:29. MatGAT: an application that generates similarity/identity matrices using protein or DNA sequences). Minor manual editing may be performed to optimise alignment between conserved motifs, as would be apparent to a person skilled in the art. Furthermore, instead of using full-length sequences for the identification of homologues, specific domains may also be used. The sequence identity values may be determined over the entire nucleic acid or amino acid sequence or over selected domains or conserved motif(s), using the programs mentioned above using the default parameters. For local alignments, the Smith-Waterman algorithm is particularly useful (Smith T F, Waterman M S (1981) J. Mol. Biol 147(1); 195-7).
Furthermore, PATL polypeptides typically have Phosphatidylinositide (PtdIns) and/or Phosphatidylcholine (PtdCho) binding activity. Preferably they bind Phosphatidylinositol. Phosphatidylinositol refers to any glycophospholipid with its sn-glycerol 3-phosphate residue esterified to the 1-hydroxyl group of 1 D-myo-inositol. Alternatively PATL polypeptides typically catalyze the transfer of Phosphatidylinosito and Phosphatidylcholine between membranes in vitro. Methods to assay PtdIns/PtdCho binding and or transfer through membranes are well know in the art (Bankaitis V A, et al. (1990). Nature 347: 561-2; Peterman et al. (2004)).
Alternatively, PATL nucleic acids may be identified in a functional complementation test by their ability to complement the defective growth phenotype of Saccharomyces cerevisie sec14 temperature sensitive mutants. In this in vivo, test the PATL nucleic acids are capable of expressing a PATL polypeptide in yeast. Several S. cerevisie sec14 temperature sensitive mutant strains and methods for their complementation are well known in the art (Kearns et al. (1998) EMBO J. 17, 4004-17; Kapranov et al. (2001); Lee et al. (2000) Biochym biophys Acta 1486:55-71).
In addition, PATL polypeptides, PRP38 polypeptides, GATA-like polypeptides, or ADA2 polypeptides, when expressed in rice and evaluated according to the methods of the present invention as outlined in Examples 7 and 8, give plants having enhanced yield related traits, in particular any one or more of total seed weight, seed filling rate, number of filled seeds and total number of flowers per panicle.
The present invention is illustrated by transforming plants with the nucleic acid sequence represented by SEQ ID NO: 1, encoding the polypeptide sequence of SEQ ID NO: 2, or SEQ ID NO: 76, encoding the polypeptide sequence of SEQ ID NO: 77, or SEQ ID NO: 128, encoding the polypeptide sequence of SEQ ID NO: 129, or SEQ ID NO: 181, encoding the polypeptide sequence of SEQ ID NO: 182, or SEQ ID NO: 215, encoding the WDR23-like polypeptide sequence of SEQ ID NO: 216. However, performance of the invention is not restricted to these sequences; the methods of the invention may advantageously be performed using any PATL-encoding nucleic acid or PATL polypeptide, PRP38-encoding nucleic acid or PRP38 polypeptide, or GATA-like-encoding nucleic acid or GATA-like polypeptide, or ADA2-encoding nucleic acid or ADA2 polypeptide, or a WDR23-like polypeptide as defined herein.
Examples of nucleic acids encoding PATL polypeptides, or PRP38 polypeptides, or GATA-like polypeptides, or ADA2 polypeptides, or WDR23-like polypeptides are given in Table A of Example 1 herein. Such nucleic acids are useful in performing the methods of the invention. The amino acid sequences given in Table A of Example 1 are example sequences of orthologues and paralogues of the PATL polypeptide represented by SEQ ID NO: 2, or of the the PRP38 polypeptide represented by SEQ ID NO: 77 or of the GATA-like polypeptide represented by SEQ ID NO: 129, or of the ADA2 polypeptide represented by SEQ ID NO: 182, or of the WDR23-like polypeptide represented by SEQ ID NO: 216, the terms “orthologues” and “paralogues” being as defined herein. Further orthologues and paralogues may readily be identified by performing a so-called reciprocal blast search. Typically, this involves a first BLAST involving BLASTing a query sequence (for example using any of the sequences listed in Table A of Example 1) against any sequence database, such as the publicly available NCBI database. BLASTN or TBLASTX (using standard default values) are generally used when starting from a nucleotide sequence, and BLASTP or TBLASTN (using standard default values) when starting from a protein sequence. The BLAST results may optionally be filtered. The full-length sequences of either the filtered results or non-filtered results are then BLASTed back (second BLAST) against sequences from the organism from which the query sequence is derived (where the query sequence is SEQ ID NO: 1 or SEQ ID NO: 2, the second BLAST would therefore be against Oryza sativa sequences; or where the query sequence is SEQ ID NO: 76 or SEQ ID NO: 77, SEQ ID NO: 128 or SEQ ID NO: 129, SEQ ID NO: 181 or SEQ ID NO: 182, or SEQ ID NO: 215 or SEQ ID NO: 216, the second BLAST would therefore be against Arabidopsis thaliana sequences). The results of the first and second BLASTs are then compared. A paralogue is identified if a high-ranking hit from the first blast is from the same species as from which the query sequence is derived, a BLAST back then ideally results in the query sequence amongst the highest hits; an orthologue is identified if a high-ranking hit in the first BLAST is not from the same species as from which the query sequence is derived, and preferably results upon BLAST back in the query sequence being among the highest hits.
PRP38 domain refers to a conserved amino acid sequence found in polypeptides that can function as pre-mRNA processing factors. It typically has about 170 amino acids in length, but is shorter in Ostta_PRP38—1 and longer in Vitvi_PRP38—1 polypeptides for example (see Table C). PRP38 domains can be represented by any one of the PRP38 domain sequences of Table C and by any polypeptide domain having in increasing order of preference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 98% or more sequence identity to any one of the PRP38 domains of Table C. PRP38 domains may comprise one or more highly conserved sequence motifs as represented by SEQ ID NO: 123 and SEQ ID NO: 124.
Alternatively, a PRP38 domain may be defined as any polypeptide domain significantly aligning to a known PRP38 polypeptide, preferably to any one of the polypeptides of Table A. A significant alignment between two polypeptides or two domains as defined herein is an alignment having in increasing order of preference an e-value lower than e−5 (e to the minus 5), 1.e-10, 1.e−15, 1.e−20, 1.e−25, 1.e−50, 1.e−75, 1.e−100, 1.e−200, 1.e−300, 1.e−400, 1.e−500, 1.e−600, 1.e−700 and 1.e−800.
The polypeptide sequences may be aligned using any of the methods well known in the art, including global and local alignment methods such as Blast algorithms, e.g. the algorithm described in Altschul, S F, et al. (1990) J. Mol. Biol. 215:403-10. The probability for the alignment to occur with a given sequence is taken as basis for identifying similar polypeptides. A parameter that is typically used to represent such probability is called an e-value (E-value). The E-value is a measure of the reliability of the S score. The S score is a measure of the similarity of the query sequence to the known sequence comprising the PRP38 domain. The e-value describes how often a given S score is expected to occur at random. The e-value cut-off may be as high as 1.0.
Preferably PRP38 polypeptides useful in the methods of the invention comprise a PRP38 domain having in increasing order of preference an e-value lower than e-5, 1.e-10, 1.e−15, 1.e−20, 1. e−25, 1.e−50, 1.e−75, 1.e−100, 1.e−200, 1.e−300, 1.e−400, 1.e−500, 1.e−600, 1.e−700 and 1.e−800 when aligned with a known PRP38 domain, more preferably when aligned to any one of the PRP38 domains of Table C.
A PRP38 domain occurring in a polypeptide may be identified by searching specialized databases containing collections of multiple sequence alignments and hidden Markov models covering conserved protein domains and families, such as Pfam which available at the Sanger Institute, United Kingdom. Example 2 herein shows the results of a Pfam search using PRP38 polypeptides as query sequence.
A DUF1777 domain is a conserved amino acid sequence found in a small subset of the proteins described to date. It is typically 140 to 150 amino acids in length, although much shorter versions are also found, see for example the DUF1777 domain of the Chlre_PRP38—1 polypeptide (Table C). DUF1777 domains have been identified in different living organism based on sequence homology. A compilation of DUF domains can be found in the Pfam database available at the Sanger Institute (UK). The presence of a DUF1777 domain in a polypeptide may be readily identified by searching specialized databases containing collections of multiple sequence alignments and hidden Markov models covering conserved protein domains and families, such as Pfam. A polypeptide comprising a DUF1777 domain will register as a hit with a previously known DUF1777 domain in a search in Pfam database. The amino acid sequences of DUF1777 domains in proteins of eukaryotic origin are often largely divergent, with only a few amino acid residues precisely conserved. Typically the e-value of an alignment of two DUF1777 domains is high, typically above 0.001, more typically above 0.01.
Preferably PRP38 polypeptides useful in the methods of the invention comprise a DUF1777 domain having in increasing order of preference an e-value lower than 1.e−5, 1.e-10, 1.e−15, 1.e−20, 1.e−25, 1.e−50, 1.e−75, 1.e−100, 1.e−200, 1.e−300, 1.e−400, 1.e−500, 1.e−600, 1.e−700 and 1.e−800 when aligned with a known DUF1777 domain, more preferably when aligned to any one of the DUF1777 domains of Table C.
More preferably, PRP38 polypeptides useful in the methods of the invention comprise a DUF1777 having in increasing order of preference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 98% or more sequence identity to any one of the DUF1777 domains of Table C.
A DUF1777 domain occurring in a polypeptide may be identified by searching specialized databases containing collections of multiple sequence alignments and hidden Markov models covering conserved protein domains and families, such as Pfam available at the Sanger Institute, United Kingdom. Example 2 herein shows the results of a Pfam search using PRP38 polypeptides as query sequence
PRP38 polypeptides may comprise an RS domain. An RS domain is a polypeptide region rich in Arginine (R) and Serine (S) amino acid residues. RS domains comprise a multiplicity of dipeptides as represented by the sequence RS (Arginine-Serine), RE (Arginine-Glutamic acid), RD (Arginine-Aspartic acid). Typically the dipeptides are spread over a region of the PRP38 polypeptide extending from 4 to 150 amino acids. The dipeptides abovementioned may occur in clusters, such clusters composed exclusively of any one or more of the RS, RE, RD dipeptides and being typically between 4 and 40 amino acids long.
Preferably, the PRP38 polypeptides useful in the methods of the invention comprise one or more of the dipeptides RS, RE, RD in a stretch of 4, 6, 8, 10, 12, 14, 20, 24, 30, 40, 50, 100 or more until a maximum of 150 amino acids.
More preferably, the PRP38 polypeptides useful in the methods of the invention comprise two or more dipeptide clusters having exclusively RS, RE and/or RD amino acid residues. Further preferably, the PRP38 polypeptide comprises 4 dipeptide clusters, most preferably the dipeptide cluster represented by RSRSRSRS (SEQ ID NO: 287).
PRP38 polypeptides may additionally comprise any one or more of the following conserved sequence motifs:
- (iv) SEQ ID NO: 120/Motif Ib: RRPPSVKASLSVSFGQRAPHRASTRDSSPVRRT, or a motif having in increasing order of preference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity to Motif Ib; and/or
- (v) SEQ ID NO: 121/Motif IIb: SPYIRA(I/V)GFLYLRY, or a motif having in increasing order of preference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity to Motif IIb; and/or
- (vi) SEQ ID NO: 122/Motif IIIb: KLKDLYGD, or a motif having in increasing order of preference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more sequence identity to Motif IIIb.
Preferably, PRP38 polypeptides useful in the methods of the invention comprise any one or more of the following motifs:
- (i) Motif 1 b (SEQ ID NO: 120) wherein any amino acid residue may be substituted by a conservative amino acid and/or up to 50% of the amino acid residues may be substituted by a non conservative amino acid.
- (ii) Motif 1 b (SEQ ID NO: 121) wherein any amino acid residue may be substituted by a conservative amino acid and/or up to 50% of the amino acid residues may be substituted by a non conservative amino acid.
- (iii) Motif 1b (SEQ ID NO: 122) wherein any amino acid residue may be substituted by a conservative amino acid and/or up to 50% of the amino acid residues may be substituted by a non conservative amino acid.
The PRP38 polypeptides useful in the methods of the invention are preferably those having in increasing order of preference at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 92%, 93%, 95%, 96%; 98% or more sequence identity to any of polypeptide given in Table A.
Preferably, the PRP38 polypeptide sequence useful in the methods of the invention is one which when used in the construction of a phylogenetic tree, such as the one depicted in
Typically in PRP38 polypeptides, PRP38 domain is found at the N-terminus and DUF1777 domain at the C-terminus. PRP38 polypeptides typically have an acidic C-terminus and are localized in the nucleus.
Furthermore, PRP38 polypeptides typically have pre-mRNA splicing activity. Tools and techniques for measuring pre-mRNA splicing activity are well known in the art (Blanton S, et al. (1992) Mol Cell Biol 12(9):3939-47; Stevens S W and Abelson J (1999) Proc Natl Acad Sci USA 96(13):7226-31; Gottschalk A, et al. (1999) EMBO J 18(16):4535-48; Pandit S, et al. (2006) Proc Natl Acad Sci USA 103(37):13700-5).
In addition, PRP38 polypeptides, when expressed in rice according to the methods of the present invention as outlined in Examples 7 and 8, give plants having enhanced yield related traits, in particular any one or more of above ground leaf biomass, emergence vigour, total seed weight, seed filling rate, harvest index, and total number of seeds.
The present invention is illustrated by transforming plants with the nucleic acid sequence represented by SEQ ID NO: 76, encoding the polypeptide sequence of SEQ ID NO: 77. However, performance of the invention is not restricted to these sequences; the methods of the invention may advantageously be performed using any PRP38-encoding nucleic acid or PRP38 polypeptide as defined herein.
A “GATA-like polypeptide” as defined herein refers to any Zn finger transcription factor comprising a GATA domain (as defined in for example the SMART database under accession SM00401). Preferably, the “GATA-like polypeptide” useful in the methods of the present invention comprises a single GATA domain with 18 or 20 amino acids between the second and the third Cys residue of the Zn finger, more preferably there are 18 amino acids between the second and the third Cys residue of the Zn finger (CX2CX18CX2C) (SEQ ID NO: 288). The term “zinc finger” or “Zn finger” is known in the art and refers to the sequence motif in which cysteines and/or histidines coordinate a zinc atom to form local peptide structures that are required for specific functions.
Further preferably, the GATA-like polypeptide useful in the methods of the present invention belongs to subfamily II as defined by Reyes et al. (Plant Physiol. 134, 1718-1732, 2004). Subfamily II GATA transcription factors typically consists of genes with 2 or 3 exons, where the zinc finger has been split between the 2 last exons. Subfamily II GATA transcription factors also has 18 residues in the Zn finger loop.
The GATA domain in the GATA-like polypeptide useful in the methods of the present invention preferably comprises a Class B type Zn finger as defined in Reyes et al. (2004). Class B Zn finger domains have 18 residues in the Zn finger loop, and are further characterised by the presence of a conserved Ser residue (in position 27 of the GATA domain shown in
Preferably, the GATA domain comprises motif 1c and/or motif 2c:
Preferably, the GATA-like polypeptide useful in the methods of the present invention also comprises motif 3c (SEQ ID NO: 132):
(A/S) (A/w)X(L/C) (L/N) (M/L/V) (T/L/A) (L/D) (S/R)wherein X can be any amino acid, preferably X is one of M, L, V, I, R.
Alternatively, the homologue of a GATA-like protein has in increasing order of preference at least 14%, 15%, 20%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% overall sequence identity to the amino acid represented by SEQ ID NO: 129, provided that the homologous protein comprises the GATA domain as defined above and one or more of the conserved motifs of above. The overall sequence identity is determined using a global alignment algorithm, such as the Needleman Wunsch algorithm in the program GAP (GCG
Wisconsin Package, Accelrys), preferably with default parameters. Compared to overall sequence identity, the sequence identity will generally be higher when only conserved domains or motifs are considered, such as the GATA domain.
Preferably, the polypeptide sequence which when used in the construction of a phylogenetic tree, such as the one depicted in
Furthermore, GATA-like polypeptides (at least in their native form) typically have DNA-binding activity activity. The consensus DNA sequence that is recognised by the GATA-Zn finger is [AT]GATA[AG]. Tools and techniques for measuring DNA binding activity are well known in the art, see for example Teakle et al. (Plant Mol. Biol. 50, 43-57, 2002) or Ghirlando and Trainor (J. Biol. Chem. 278, 45620-45628, 2003).
In addition, GATA-like polypeptides, when expressed in rice according to the methods of the present invention as outlined in Examples 7 and 8, give plants having increased Thousand Kernel Weight.
The present invention is illustrated by transforming plants with the nucleic acid sequence represented by SEQ ID NO: 128, encoding the polypeptide sequence of SEQ ID NO: 129.
However, performance of the invention is not restricted to these sequences; the methods of the invention may advantageously be performed using any GATA-like-encoding nucleic acid or GATA-like polypeptide as defined herein.
An “ADA2 polypeptide” as defined herein refers to any transcriptional adaptor polypeptide comprising two or more of the following motifs:
-
- (vii) a Zinc (Zn) finger ZZ type domain
- (viii) a SANT DNA binding domain
- (ix) a Calcium EF hand domain
- (x) a SWIRM domain
A prototype of Zinc (Zn) finger ZZ type domain is present in dystrophin, CBP/p300 protein. The Cys-×2-Cys motifs in ZZ domains are reminiscent of the Cys-×2-Cys knuckles that occur in zinc fingers. Four to six cysteine residues in the domain sequence are responsible for coordinating zinc ions, to reinforce the structure (Ponting et al.
Trends Biochem Sci 1996; 21:11-13). The Zinc finger ZZ type domain in ADA2 polypeptides could be involved in facilitating protein-protein interactions. A fragment of Arabidopsis thaliana ADA2a and ADA2b proteins comprising the ZZ zinc finger domain has been shown to bind to GCN5 protein (Mao et al 2006).
A SANT DNA binding domain is a subfamily of the Myb DNA binding domain (Aasland et al. 1996 Trends Biochem Sci 1996; 21:87-88). Polypeptides comprising SANT DNA binding domains specifically recognize the sequence YAAC(G/T)G present in a gene promoter. Preferred ADA polypeptides useful in the methods of the invention bind to a gene promoter comprising the sequence YAAC(G/T)G where Y can be C or T).
The SWIRM domain (Pfam accession number PF04433) is a small alpha-helical domain of about 85 amino acid residues found in eukaryotic chromosomal proteins. It is named after the proteins SWI3, RSC8 and MOIRA in which it was first recognized. This domain is predicted to mediate protein-protein interactions in the assembly of chromatin-protein complexes Lenkart et al. Proc Natl Acad Sci USA. 2006; 103:2057-2062).
A Calcium (Ca) binding EF hand domain consists of a twelve residue loop flanked on both side by a twelve residue alpha-helical domain. In an EF-hand loop the calcium ion is coordinated in a pentagonal bipyramidal configuration. The six residues involved in the binding are in positions 1, 3, 5, 7, 9 and 12; these residues are denoted by X, Y, Z, −Y, −X and −Z. The invariant Glu or Asp at position 12 provides two oxygens for liganding Ca (bidentate ligand) (Finn and Forsen 1995 Structure, 3:7-11).
A preferred ADA2 polypeptide useful in the methods of the invention comprises two or more of the following domains:
- (i) A Zinc (Zn) finger ZZ type domain as represented by SEQ ID NO: 207: kpglyccnycdkdlsglvrfkcavcmdfdlcvecfsvgvelnrhkn or a domain having in increasing order of preference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82%, 85%, 90%, 92%, 95%, 97% or more sequence identity to the domain represented by SEQ ID NO: 207 or to any Zn finger ZZ type domain as present in any one of the polypeptides of Table A;
- (ii) A SANT DNA binding domain as represented by SEQ ID NO: 208: vtsdwnadeeillleaiatygfgnwkevadhvgsktttecikhfnsaym, or a domain having in increasing order of preference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82%, 85%, 90%, 92%, 95%, 97% or more sequence identity to the domain represented by SEQ ID NO: 208 or to any SANT domain as present in any one of the polypeptides of Table A;
- (iii) A Ca binding EF hand domain as represented by SEQ ID NO: 209: dndaeqlladmef, or a domain having in increasing order of preference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82%, 85%, 90%, 92%, 95%, 97% or more sequence identity to the domain represented by SEQ ID NO: 209 or to any Ca binding EF hand domain as present in any one of the polypeptides of Table A;
- (iv) A SWIRM domain as represented by SEQ ID NO: 210: physgldtwdydgllgadllsetekkmcnetrilpyhylkmldiltreikkgqikkksdaysffkvepskydryydmlyhkgi gdst, or a domain having in increasing order of preference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82%, 85%, 90%, 92%, 95%, 97% or more sequence identity to the domain represented by SEQ ID NO: 210 or to any SWRIM domain as present in any one of the polypeptides of Table A.
The ADA2 polypeptides useful in the methods of the invention are preferably those having in increasing order of preference at least 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 92%, 93%, 95%, 96%, 98% or more sequence identity to any of polypeptide given in Table A.
A Zinc (Zn) finger ZZ type domain, a SANT DNA binding, a Calcium EF hand domain and a SWIRM domain comprised in a polypeptide may be identified by searching specialized databases containing collections of multiple sequence alignments and hidden Markov models covering conserved protein domains and families, such as Pfam which available at the Sanger Institute, United Kingdom. Alternatively the domains abovementioned may be found by scanning The Integrated Resource of Protein Families, Domains and Sites (InterPro) database to detect a significant sequence alignment with known Zinc (Zn) finger ZZ type domain, a SANT DNA binding, a Calcium EF hand domain and a SWIRM domain. Interpro database is an integrated interface for the commonly used signature databases for text- and sequence-based searches. The InterPro database combines these databases, which use different methodologies and varying degrees of biological information about well-characterized proteins to derive protein signatures. Collaborating databases include SWISS-PROT, PROSITE, TrEMBL, PRINTS, ProDom and Pfam, Smart and TIGRFAMs. Interpro is hosted at the European Bioinformatics Institute in the United Kingdom. A significant alignment between the sequence of two polypeptides or two domains as defined herein is an alignment having in increasing order of preference an e-value lower than e−5 (e to the minus 5), 1.e-10, 1.e−151.e−20, 1.e−25, 1.e−50, 1.e−75, 1.e−100, 1.e−200, 1.e−300, 1.e−400, 1.e−500, 1.e−600, 1.e−700 and 1.e−800. The polypeptide sequences may be aligned using any of the methods well known in the art, including global and local alignment methods such as Blast algorithms, e.g. the algorithm described in Altschul, S F, et al. (1990) J. Mol. Biol. 215:403-10. The probability for the alignment to occur with a given sequence is taken as basis for identifying similar polypeptides. A parameter that is typically used to represent such probability is called an e-value (E-value). The E-value is a measure of the reliability of the S score. The S score is a measure of the similarity of the two sequenced being aligned. The e-value describes how often a given S score is expected to occur at random. The e-value may be as high as 1.0.
Preferably ADA2 polypeptides useful in the methods of the invention comprise two or more of the following domains:
- (i) A Zinc (Zn) finger ZZ type domain having in increasing order of preference an e-value lower than e-5, 1.e-10, 1.e-15, 1.e-20, 1.e-25, 1.e-50, 1.e-75, 1.e-100, 1.e-200, 1.e-300, 1.e-400, 1.e-500, 1.e-600, 1.e-700 and 1.e-800 when aligned with a known Zn finger ZZ type domain, more preferably when aligned to any one of the Zn finger ZZ type of Table C;
- (ii) A SANT DNA binding domain having in increasing order of preference an e-value lower than e-5, 1.e-10, 1.e-15, 1.e-20, 1.e-25, 1.e-50, 1.e-75, 1.e-100, 1.e-200, 1.e-300, 1.e-400, 1.e-500, 1.e-600, 1.e-700 and 1.e-800 when aligned with a known SANT DNA binding domain, more preferably when aligned to any one of the SANT DNA binding domain domains of Table C;
- (iii) A Calcium EF hand domain having in increasing order of preference an e-value lower than e-5, 1.e-10, 1.e-15, 1.e-20, 1.e-25, 1.e-50, 1.e-75, 1.e-100, 1.e-200, 1.e-300, 1.e-400, 1.e-500, 1.e-600, 1.e-700 and 1.e-800 when aligned with a known Calcium EF hand domain, more preferably when aligned to any one of the Calcium EF hand domains of Table C.
- (iv) A SWIRM domain having in increasing order of preference an e-value lower than e-5, 1.e-10, 1.e-15, 1.e-20, 1.e-25, 1.e-50, 1.e-75, 1.e-100, 1.e-200, 1.e-300, 1.e-400, 1.e-500, 1.e-600, 1.e-700 and 1.e-800 when aligned with a known SWIRM domain domain, more preferably when aligned to any one of the SWIRM domain domains of Table C.
Alternatively, an ADA2 polypeptide may be defined as any polypeptide significantly aligning to a known ADA2 polypeptide, preferably to any one of the polypeptides of Table A.
Preferably, the ADA2 polypeptide sequence useful in the methods of the invention is one which when used in the construction of a phylogenetic tree, such as the one depicted in
Typically in ADA2 polypeptides, Zn Finger ZZ domain is found at the N-terminus, the SANT-DNA binding domain in the central portion and the SWIRM domain is found at the C-terminus.
ADA2 polypeptides and typically localized in the nucleus of the cell. The plant ADA2 polypeptides may comprise a nuclear localization signal as for example that shown in
Furthermore, ADA2 polypeptides typically enhance the ability of GCN5 to acetylate histones in vitro and enable GCN5 to acetylate nucleosomal histones. Tools and techniques for measuring histone acetylation have been previously described (Stockinger et al 2001; Mao et al. 2006). Regulation of the activity of ADA2 polypeptides of plant origin in plant cells may occur via acetylation. It has been proposed that lysine residues K257 and K215 in ADA2a in ADA2b of Arabidopsis thalina could be acetylated.
The invention provides nucleic acid sequences encoding WDR23-like polypeptides and WDR23-like polypeptides, whereby increased expression of the isolated nucleic acid sequences in plants increase yield-related traits relative to control plants.
According to one embodiment of the present invention, there is therefore provided an isolated nucleic acid sequence encoding a WDR23-like polypeptide comprising:
-
- (i) an isolated nucleic acid sequence as represented by SEQ ID NO: 219, SEQ ID NO: 225, or SEQ ID NO: 229;
- (ii) the complement of an isolated nucleic acid sequence as represented by SEQ ID NO: 219, SEQ ID NO: 225, or SEQ ID NO: 229;
- (iii) an isolated nucleic acid sequence encoding a polypeptide sequence as represented by SEQ ID NO: 220, SEQ ID NO: 226, or SEQ ID NO: 230;
- (iv) an isolated nucleic acid sequence deduced from a polypeptide sequence as represented by SEQ ID NO: 220, SEQ ID NO: 226, or SEQ ID NO: 230, as a result of the degeneracy of the genetic code;
- (v) an isolated nucleic acid sequence capable of stringently hybridising to a nucleic acid sequence as represented by SEQ ID NO: 219, SEQ ID NO: 225, or SEQ ID NO: 229, or to its complement;
- (vi) an isolated nucleic acid sequence which encodes a polypeptide having in increasing order of preference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity with a polypeptide sequence as represented by SEQ ID NO: 220, SEQ ID NO: 226, or SEQ ID NO: 230;
- (iv) an isolated nucleic acid sequence which encodes a polypeptide comprising a domain having, in increasing order of preference, at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity with a Conserved Domain as represented by SEQ ID NO: 271.
According to a further embodiment of the present invention, there is also provided an isolated WDR23-like polypeptide comprising:
-
- (i) a polypeptide sequence represented by SEQ ID NO: 220, SEQ ID NO: 226, or SEQ ID NO: 230;
- (ii) a polypeptide sequence having, in increasing order of preference, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% amino acid sequence identity with a polypeptide sequence as represented by SEQ ID NO: 220, SEQ ID NO: 226, or SEQ ID NO: 230;
- (iii) a polypeptide comprising a domain having, in increasing order of preference, at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity with a Conserved Domain as represented by SEQ ID NO: 271;
- (iii) derivatives of any of the polypeptide sequences given in (i) to (iii) above.
According to a further embodiment, the present invention provides a method for increasing yield-related traits in plants relative to control plants, comprising increasing expression in a plant of a nucleic acid sequence encoding a WDR23-like polypeptide.
A preferred method for increasing expression of a nucleic acid sequence encoding a WDR23-like polypeptide is by introducing and expressing in a plant a nucleic acid sequence encoding a WDR23-like polypeptide.
Any reference hereinafter to a “polypeptide useful in the methods of the invention” is taken to mean a WDR23-like polypeptide as defined herein. Any reference hereinafter to a “nucleic acid sequence useful in the methods of the invention” is taken to mean a nucleic acid sequence capable of encoding such a WDR23-like polypeptide. The nucleic acid sequence to be introduced into a plant (and therefore useful in performing the methods of the invention) is any nucleic acid sequence encoding the type of polypeptide, which will now be described, hereafter also named “WDR23-like nucleic acid sequence” or “WDR23-like gene”.
A “WDR23-like polypeptide” as defined herein refers to any polypeptide comprising a domain having at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid sequence identity to a Conserved Domain (CD) as represented by SEQ ID NO: 271.
Alternatively or additionally, a “WDR23-like polypeptide” as defined herein refers to any polypeptide comprising: (i) at least four WD40 repeats with a PFAM accession PF00400; and
- (ii) at least two conserved DxR motifs at the end of two consecutive WD40 repeats.
Alternatively or additionally, a “WDR23-like polypeptide” as defined herein refers to any polypeptide having, in increasing order of preference, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid sequence identity to the WDR23-like polypeptide as represented by SEQ ID NO: 216 or to any of the polypeptide sequences given in Table A herein.
Analysis of the polypeptide sequence of SEQ ID NO: 216 is presented below in Example 4 herein. For example, a WDR23-like polypeptide as represented by SEQ ID NO: 216 comprises at least four WD40 repeats with a PFAM accession PF00400. Domains may also be identified using routine techniques, such as by sequence alignment. An alignment of the full length polypeptides of Table A herein, is shown in
Methods for the alignment of sequences for comparison are well known in the art, such methods include GAP, BESTFIT, BLAST, FASTA and TFASTA. GAP uses the algorithm of Needleman and Wunsch ((1970) J Mol Biol 48: 443-453) to find the global (i.e. spanning the complete sequences) alignment of two sequences that maximizes the number of matches and minimizes the number of gaps. The BLAST algorithm (Altschul et al. (1990) J Mol Biol 215: 403-10) calculates percent sequence identity and performs a statistical analysis of the similarity between the two sequences. The software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information (NCBI). Homologues may readily be identified using, for example, the ClustalW multiple sequence alignment algorithm (version 1.83), with the default pairwise alignment parameters, and a scoring method in percentage. Global percentages of similarity and identity may also be determined using one of the methods available in the MatGAT software package (Campanella et al., (2003) BMC Bioinformatics, 10: 29. MatGAT: an application that generates similarity/identity matrices using protein or DNA sequences). Minor manual editing may be performed to optimise alignment between conserved motifs, as would be apparent to a person skilled in the art. Furthermore, instead of using full-length sequences for the identification of homologues, specific domains may also be used. The sequence identity values may be determined over the entire nucleic acid sequence or polypeptide sequence or over selected domains or conserved motif(s), using the programs mentioned above using the default parameters. Example 3 herein describes in Table B the percentage identity between the WDR23-like polypeptide as represented by SEQ ID NO: 216 and the full length WDR23-like polypeptides listed in Table A, which can be as low as 54% amino acid sequence identity. The percentage identity can be increased to 69% if the identity calculation is performed between the Conserved Domain (CD) as represented by SEQ ID NO: 271 (comprised in SEQ ID NO: 216) and the Conserved Domain of the WDR23-like polypeptides of Table A and represented in
The task of protein subcellular localisation prediction is important and well studied. Knowing a protein's localisation helps elucidate its function. Experimental methods for protein localization range from immunolocalization to tagging of proteins using green fluorescent protein (GFP) or beta-glucuronidase (GUS). Such methods are accurate although labor-intensive compared with computational methods. Recently much progress has been made in computational prediction of protein localisation from sequence data. Among algorithms well known to a person skilled in the art are available at the ExPASy Proteomics tools hosted by the Swiss Institute for Bioinformatics, for example, PSort, TargetP, ChloroP, LocTree, Predotar, LipoP, MITOPROT, PATS, PTS1, SignalP, TMHMM, and others.
Furthermore, WDR23-like polypeptides useful in the methods of the present invention (at least in their native form) typically are capable of interacting with other polypeptides, through their WD40 repeat motifs. Many assays exist to protein-protein activity, such as yeast two-hybrid assays, tandem-affinity purification (TAP) followed by mass spectrometry, co-affinity purification, etc.
High-ranking hits are those having a low E-value. The lower the E-value, the more significant the score (or in other words the lower the chance that the hit was found by chance). Computation of the E-value is well known in the art. In addition to E-values, comparisons are also scored by percentage identity. Percentage identity refers to the number of identical nucleotides (or amino acids) between the two compared nucleic acid (or polypeptide) sequences over a particular length. In the case of large families, ClustalW may be used, followed by a neighbour joining tree, to help visualize clustering of related genes and to identify orthologues and paralogues.
Nucleic acid variants may also be useful in practising the methods of the invention. Examples of such variants include nucleic acids encoding homologues and derivatives of any one of the amino acid sequences given in Table A of Example 1, the terms “homologue” and “derivative” being as defined herein. Also useful in the methods of the invention are nucleic acids encoding homologues and derivatives of orthologues or paralogues of any one of the amino acid sequences given in Table A of Example 1. Homologues and derivatives useful in the methods of the present invention have substantially the same biological and functional activity as the unmodified protein from which they are derived.
Further nucleic acid variants useful in practising the methods of the invention include portions of nucleic acids encoding PATL, or PRP38, or GATA-like or ADA2, or WDR23-like polypeptides polypeptides, nucleic acids hybridising to nucleic acids encoding PATL, or PRP38, or GATA-like or ADA2, or WDR23-like polypeptides polypeptides, splice variants of nucleic acids encoding PATL, or PRP38, or GATA-like or ADA2, or WDR23-like polypeptides polypeptides, allelic variants of nucleic acids encoding PATL, or PRP38, or GATA-like or ADA2, or WDR23-like polypeptides polypeptides and variants of nucleic acids encoding PATL, or PRP38, or GATA-like or ADA2, or WDR23-like polypeptides polypeptides obtained by gene shuffling. The terms hybridising sequence, splice variant, allelic variant and gene shuffling are as described herein.
Nucleic acids encoding PATL, or PRP38, or GATA-like or ADA2, or WDR23-like polypeptides polypeptides need not be full-length nucleic acids, since performance of the methods of the invention does not rely on the use of full-length nucleic acid sequences. According to the present invention, there is provided a method for enhancing yield-related traits in plants, comprising introducing and expressing in a plant a portion of any one of the nucleic acid sequences given in Table A of Example 1, or a portion of a nucleic acid encoding an orthologue, paralogue or homologue of any of the amino acid sequences given in Table A of Example 1.
A portion of a nucleic acid may be prepared, for example, by making one or more deletions to the nucleic acid. The portions may be used in isolated form or they may be fused to other coding (or non-coding) sequences in order to, for example, produce a protein that combines several activities. When fused to other coding sequences, the resultant polypeptide produced upon translation may be bigger than that predicted for the protein portion.
Portions useful in the methods of the invention, encode a PATL, or PRP38, or ADA2 polypeptide as defined herein, and have substantially the same biological activity as the amino acid sequences given in Table A of Example 1. Portions useful in the methods of the invention comprise a protein domain having in increasing order of preference 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 92%, 93%, 95%, 96%; 98% or more sequence identity to any one of the conserved domains defined in Table C. Preferably, the portion is a portion of any one of the nucleic acids given in Table A of Example 1, or is a portion of a nucleic acid encoding an orthologue or paralogue of any one of the amino acid sequences given in Table A of Example 1. Preferably the portion is at least 50, 100, 150, 200, 300, 400, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1200 or more consecutive nucleotides in length, the consecutive nucleotides being of any one of the nucleic acid sequences given in Table A of Example 1, or of a nucleic acid encoding an orthologue or paralogue of any one of the amino acid sequences given in Table A of Example 1. Preferably, the portion encodes a fragment of an amino acid sequence which, when used in the construction of a phylogenetic tree, such as the one depicted in
Concerning GATA-like polypeptides, portions useful in the methods of the invention, encode a GATA-like polypeptide as defined herein, and have substantially the same biological activity as the amino acid sequences given in Table A of Example 1. Preferably, the portion is a portion of any one of the nucleic acids given in Table A of Example 1, or is a portion of a nucleic acid encoding an orthologue or paralogue of any one of the amino acid sequences given in Table A of Example 1. Preferably the portion is at least 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050 consecutive nucleotides in length, the consecutive nucleotides being of any one of the nucleic acid sequences given in Table A of Example 1, or of a nucleic acid encoding an orthologue or paralogue of any one of the amino acid sequences given in Table A of Example 1. Most preferably the portion is a portion of the nucleic acid of SEQ ID NO: 128. Preferably, the portion encodes a fragment of an amino acid sequence which, when used in the construction of a phylogenetic tree, such as the one depicted in
Portions useful in the methods of the invention, encode a WDR23-like polypeptide as defined herein, and have substantially the same biological activity as the polypeptide sequences given in Table A of Example 1. Preferably, the portion is a portion of any one of the nucleic acid sequences given in Table A of Example 1, or is a portion of a nucleic acid sequence encoding an orthologue or paralogue of any one of the polypeptide sequences given in Table A of Example 1. Preferably the portion is, in increasing order of preference at least 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1480 or more consecutive nucleotides in length, the consecutive nucleotides being of any one of the nucleic acid sequences given in Table A of Example 1, or of a nucleic acid sequence encoding an orthologue or paralogue of any one of the polypeptide sequences given in Table A of Example 1. Preferably, the portion is a portion of a nucleic sequence encoding a polypeptide sequence polypeptide comprising a domain having at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid sequence identity to a Conserved Domain (CD) as represented by SEQ ID NO: 271. Most preferably, the portion is a portion of the nucleic acid sequence of SEQ ID NO: 215.
Another nucleic acid variant useful in the methods of the invention is a nucleic acid capable of hybridising, under reduced stringency conditions, preferably under stringent conditions, with a nucleic acid encoding a PATL, or PRP38, or GATA-like, or ADA2, or WDR23-like polypeptide as defined herein, or with a portion there of as defined herein.
According to the present invention, there is provided a method for enhancing yield-related traits in plants, comprising introducing and expressing in a plant a nucleic acid capable of hybridizing to any one of the nucleic acids given in Table A of Example 1, or comprising introducing and expressing in a plant a nucleic acid capable of hybridising to a nucleic acid encoding an orthologue, paralogue or homologue of any of the nucleic acid sequences given in Table A of Example 1.
Hybridising sequences useful in the methods of the invention encode a PATL, or PRP38, or GATA-like, or ADA2, or WDR23-like polypeptide as defined herein, having substantially the same biological activity as the amino acid sequences given in Table A of Example 1. Preferably, the hybridising sequence is capable of hybridising to any one of the nucleic acids given in Table A of Example 1, or to a portion of any of these sequences, a portion being as defined above, or the hybridising sequence is capable of hybridising to a nucleic acid encoding an orthologue or paralogue of any one of the amino acid sequences given in Table A of Example 1. Most preferably, the hybridising sequence is capable of hybridising to a nucleic acid as represented by SEQ ID NO: 1 or to a portion thereof or to SEQ ID NO: 76 or to a portion thereof, or to SEQ ID NO: 128 or to a portion thereof, or to SEQ ID NO: 181 or to a portion thereof. Concerning the WDR23-like sequences, preferably, the hybridising sequence is capable of hybridising to a nucleic acid sequence encoding a polypeptide sequence comprising a domain having at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid sequence identity to a Conserved Domain (CD) as represented by SEQ ID NO: 271. Most preferably, the hybridising sequence is capable of hybridising to a nucleic acid sequence as represented by SEQ ID NO: 215 or to a portion thereof.
Preferably, the hybridising sequence encodes a polypeptide with an amino acid sequence which, when full-length and used in the construction of a phylogenetic tree, such as the one depicted in
Another nucleic acid variant useful in the methods of the invention is a splice variant encoding a PATL, or PRP38, or GATA-like, or ADA2, or WDR23-like polypeptide as defined hereinabove, a splice variant being as defined herein.
According to the present invention, there is provided a method for enhancing yield-related traits in plants, comprising introducing and expressing in a plant a splice variant of any one of the nucleic acid sequences given in Table A of Example 1, or a splice variant of a nucleic acid encoding an orthologue, paralogue or homologue of any of the amino acid sequences given in Table A of Example 1.
Preferred splice variants are splice variants of a nucleic acid represented by SEQ ID NO: 1, or a splice variant of a nucleic acid encoding an orthologue or paralogue of SEQ ID NO: 2. More preferably the splice variant is a variant of SEQ ID NO: 1. Preferably, the amino acid sequence encoded by the splice variant, when used in the construction of a phylogenetic tree, such as the one depicted in
Other preferred splice variants are splice variants of a nucleic acid represented by SEQ ID NO: 76, or a splice variant of a nucleic acid encoding an orthologue or paralogue of SEQ ID NO: 77. More preferably the splice variant is a variant of SEQ ID NO: 76, most preferably is a splice variant as represented by SEQ ID NO: 80. Preferably, the amino acid sequence encoded by the splice variant, when used in the construction of a phylogenetic tree, such as the one depicted in
More preferred splice variants are splice variants of a nucleic acid represented by SEQ ID NO: 128, or a splice variant of a nucleic acid encoding an orthologue or paralogue of SEQ ID NO: 129. Preferably, the amino acid sequence encoded by the splice variant, when used in the construction of a phylogenetic tree, such as the one depicted in
Further preferred splice variants are splice variants of a nucleic acid represented by SEQ ID NO: 181, or a splice variant of a nucleic acid encoding an orthologue or paralogue of SEQ ID NO: 182. More preferably the splice variant is a variant of SEQ ID NO: 181. Preferably, the amino acid sequence encoded by the splice variant, when used in the construction of a phylogenetic tree, such as the one depicted in
The human ortholog WRD23 is present as multiple isoforms (NCBI accession AK057636.1), so that splice variants are also predicted for plant nucleic acid sequences encoding WRD23-like polypeptides.
Concerning the WDR23-like sequences, preferred splice variants are splice variants of a nucleic acid sequence represented by SEQ ID NO: 215, or a splice variant of a nucleic acid sequence encoding an orthologue or paralogue of SEQ ID NO: 216. Preferably, the splice variant is a splice variant of a nucleic acid sequence encoding a polypeptide sequence comprising a domain having at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid sequence identity to a Conserved Domain (CD) as represented by SEQ ID NO: 271.
Another nucleic acid variant useful in performing the methods of the invention is an allelic variant of a nucleic acid encoding a PATL, or PRP38, or GATA-like, or ADA2, or WDR23-like polypeptide as defined hereinabove, an allelic variant being as defined herein.
According to the present invention, there is provided a method for enhancing yield-related traits in plants, comprising introducing and expressing in a plant an allelic variant of any one of the nucleic acids given in Table A of Example 1, or comprising introducing and expressing in a plant an allelic variant of a nucleic acid encoding an orthologue, paralogue or homologue of any of the amino acid sequences given in Table A of Example 1.
The allelic variants useful in the methods of the present invention have substantially the same biological activity as the PATL polypeptide of SEQ ID NO: 2 and any of the amino acids depicted in Table A of Example 1. Allelic variants exist in nature, and encompassed within the methods of the present invention is the use of these natural alleles. Preferably, the allelic variant is an allelic variant of SEQ ID NO: 1 or an allelic variant of a nucleic acid encoding an orthologue or paralogue of SEQ ID NO: 2. Preferably, the amino acid sequence encoded by the allelic variant, when used in the construction of a phylogenetic tree, such as the one depicted in
Other allelic variants useful in the methods of the present invention have substantially the same biological activity as the PRP38 polypeptide of SEQ ID NO: 77 and any of the amino acids depicted in Table A of Example 1. Allelic variants exist in nature, and encompassed within the methods of the present invention is the use of these natural alleles. Preferably, the allelic variant is an allelic variant of SEQ ID NO: 76 or an allelic variant of a nucleic acid encoding an orthologue or paralogue of SEQ ID NO: 77. Preferably, the amino acid sequence encoded by the allelic variant, when used in the construction of a phylogenetic tree, such as the one depicted in
Concerning the GATA-like polypeptides, the allelic variants useful in the methods of the present invention have substantially the same biological activity as the GATA-like polypeptide of SEQ ID NO: 129 and any of the amino acids depicted in Table A of Example 1. Allelic variants exist in nature, and encompassed within the methods of the present invention is the use of these natural alleles.
Preferably, the allelic variant is an allelic variant of SEQ ID NO: 128 or an allelic variant of a nucleic acid encoding an orthologue or paralogue of SEQ ID NO: 129. Preferably, the amino acid sequence encoded by the allelic variant, when used in the construction of a phylogenetic tree, such as the one depicted in
Concerning ADA2 polypeptides, the allelic variants useful in the methods of the present invention have substantially the same biological activity as the ADA2 polypeptide of SEQ ID NO: 182 and any of the amino acids depicted in Table A of Example 1. Allelic variants exist in nature, and encompassed within the methods of the present invention is the use of these natural alleles. Preferably, the allelic variant is an allelic variant of SEQ ID NO: 181 or an allelic variant of a nucleic acid encoding an orthologue or paralogue of SEQ ID NO: 182. Preferably, the amino acid sequence encoded by the allelic variant, when used in the construction of a phylogenetic tree, such as the one depicted in
Concerning WDR23-like polypeptides, the allelic variants useful in the methods of the present invention have substantially the same biological activity as the WDR23-like polypeptide of SEQ ID NO: 216 and any of the polypeptide sequences depicted in Table A of Example 1. Allelic variants exist in nature, and encompassed within the methods of the present invention is the use of these natural alleles. Preferably, the allelic variant is an allelic variant of SEQ ID NO: 215 or an allelic variant of a nucleic acid sequence encoding an orthologue or paralogue of SEQ ID NO: 216. Preferably, the allelic variant is an allelic variant of a polypeptide sequence comprising a domain having at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid sequence identity to a Conserved Domain (CD) as represented by SEQ ID NO: 271.
Gene shuffling or directed evolution may also be used to generate variants of nucleic acids encoding PATL, or PRP38, or GATA-like, or ADA2, or WDR23-like polypeptides as defined above; the term “gene shuffling” being as defined herein.
According to the present invention, there is provided a method for enhancing yield-related traits in plants, comprising introducing and expressing in a plant a variant of any one of the nucleic acid sequences given in Table A of Example 1, or comprising introducing and expressing in a plant a variant of a nucleic acid encoding an orthologue, paralogue or homologue of any of the amino acid sequences given in Table A of Example 1, which variant nucleic acid is obtained by gene shuffling.
Preferably, the amino acid sequence encoded by the variant nucleic acid obtained by gene shuffling, when used in the construction of a phylogenetic tree such as the one depicted in
Furthermore, nucleic acid variants may also be obtained by site-directed mutagenesis. Several methods are available to achieve site-directed mutagenesis, the most common being PCR based methods (Current Protocols in Molecular Biology. Wiley Eds.).
Nucleic acids encoding PATL, or PRP38, or GATA-like, or ADA2 polypeptides may be derived from any natural or artificial source. The nucleic acid may be modified from its native form in composition and/or genomic environment through deliberate human manipulation. Preferably the PATL, or the GATA-like polypeptide-encoding nucleic acid is from a plant, further preferably from a monocotyledonous plant, more preferably from the family poaceae, most preferably the nucleic acid is from Oryza sativa. Preferably the PRP38 polypeptide-encoding nucleic acid is from a plant, further preferably from a dicotyledonous plant, more preferably from the family brasicaceae, most preferably the nucleic acid is from Arabidopsis thaliana.
Preferably the ADA2 polypeptide-encoding nucleic acid is from a plant, further preferably from a dicotyledonous plant, more preferably from the family brasicaceae, most preferably the nucleic acid is from Arabidopsis thaliana. Nucleic acid sequences encoding WDR23-like polypeptides may be derived from any natural or artificial source. The nucleic acid sequence may be modified from its native form in composition and/or genomic environment through deliberate human manipulation. The nucleic acid sequence encoding a WDR23-like polypeptide is from the Eukaryota domain, preferably from the plant kingdom, further preferably from a dicotyledon plant. More preferably, the nucleic acid sequence encoding a WDR23-like polypeptide is from the Brassicaceae family, most preferably, the nucleic acid sequence is encoding a WDR23-like polypeptide is from Arabidopsis thaliana.
Advantageously, the present invention provides hitherto unknown PATL nucleic acids and polypeptide sequences.
According to a further embodiment of the present invention, there is provided an isolated nucleic acid molecule comprising:
- (i) a nucleic acid represented by SEQ ID NO: 9; SEQ ID NO: 11; SEQ ID NO: 13; SEQ ID NO: 15; SEQ ID NO: 17; SEQ ID NO: 19; SEQ ID NO: 21; SEQ ID NO: 23; SEQ ID NO: 25 and SEQ ID NO: 27;
- (ii) a nucleic acid or fragment thereof that is complementary to any one of the SEQ ID NOs given in (i);
- (iii) a nucleic acid encoding a PATL polypeptide having, in increasing order of preference, at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any one of the amino acid sequences given in SEQ ID NO: 10; SEQ ID NO: 12; SEQ ID NO: 14; SEQ ID NO: 16; SEQ ID NO: 18; SEQ ID NO: 20; SEQ ID NO: 22; SEQ ID NO: 24; SEQ ID NO: 26 and SEQ ID NO: 28;
- (iv) a nucleic acid capable of hybridizing under stringent conditions to any one of the nucleic acids given in (i), (ii) or (iii) above.
According to a further embodiment of the present invention, there is therefore provided an isolated polypeptide comprising:
- (i) an amino acid sequence having, in increasing order of preference, at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of the amino acid sequences given in SEQ ID NO: 10; SEQ ID NO: 12; SEQ ID NO: 14; SEQ ID NO: 16; SEQ ID NO: 18; SEQ ID NO: 20; SEQ ID NO: 22; SEQ ID NO: 24; SEQ ID NO: 26 and SEQ ID NO: 28;
- (ii) derivatives of any of the amino acid sequences given in (i).
Performance of the methods of the invention gives plants having enhanced yield-related traits. In particular performance of the methods of the invention gives plants having increased yield, especially increased seed yield relative to control plants. The terms “yield” and “seed yield” are described in more detail in the “definitions” section herein.
Reference herein to enhanced yield-related traits is taken to mean an increase in biomass (weight) of one or more parts of a plant, which may include aboveground (harvestable) parts and/or (harvestable) parts below ground. In particular, such harvestable parts are seeds, and performance of the methods of the invention results in plants having increased seed yield relative to the seed yield of control plants.
Taking corn as an example, a yield increase may be manifested as one or more of the following: increase in the number of plants established per square meter, an increase in the number of ears per plant, an increase in the number of rows, number of kernels per row, kernel weight, thousand kernel weight, ear length/diameter, increase in the seed filling rate (which is the number of filled seeds divided by the total number of seeds and multiplied by 100), among others. Taking rice as an example, a yield increase may manifest itself as an increase in one or more of the following: number of plants per square meter, number of panicles per plant, number of spikelets per panicle, number of flowers (florets) per panicle (which is expressed as a ratio of the number of filled seeds over the number of primary panicles), increase in the seed filling rate (which is the number of filled seeds divided by the total number of seeds and multiplied by 100), increase in thousand kernel weight, among others.
The present invention provides a method for increasing yield, especially seed yield of plants, relative to control plants, which method comprises modulating expression in a plant of a nucleic acid encoding a PATL, or PRP38, or a GATA-like, or ADA2, or a WDR23-like polypeptide as defined herein.
Since the transgenic plants according to the present invention have increased yield, it is likely that these plants exhibit an increased growth rate (during at least part of their life cycle), relative to the growth rate of control plants at a corresponding stage in their life cycle.
The increased growth rate may be specific to one or more parts of a plant (including seeds), or may be throughout substantially the whole plant. Plants having an increased growth rate may have a shorter life cycle. The life cycle of a plant may be taken to mean the time needed to grow from a dry mature seed up to the stage where the plant has produced dry mature seeds, similar to the starting material. This life cycle may be influenced by factors such as early vigour, growth rate, greenness index, flowering time and speed of seed maturation. The increase in growth rate may take place at one or more stages in the life cycle of a plant or during substantially the whole plant life cycle. Increased growth rate during the early stages in the life cycle of a plant may reflect enhanced vigour. The increase in growth rate may alter the harvest cycle of a plant allowing plants to be sown later and/or harvested sooner than would otherwise be possible (a similar effect may be obtained with earlier flowering time). If the growth rate is sufficiently increased, it may allow for the further sowing of seeds of the same plant species (for example sowing and harvesting of rice plants followed by sowing and harvesting of further rice plants all within one conventional growing period). Similarly, if the growth rate is sufficiently increased, it may allow for the further sowing of seeds of different plants species (for example the sowing and harvesting of corn plants followed by, for example, the sowing and optional harvesting of soybean, potato or any other suitable plant). Harvesting additional times from the same rootstock in the case of some crop plants may also be possible. Altering the harvest cycle of a plant may lead to an increase in annual biomass production per square meter (due to an increase in the number of times (say in a year) that any particular plant may be grown and harvested). An increase in growth rate may also allow for the cultivation of transgenic plants in a wider geographical area than their wild-type counterparts, since the territorial limitations for growing a crop are often determined by adverse environmental conditions either at the time of planting (early season) or at the time of harvesting (late season). Such adverse conditions may be avoided if the harvest cycle is shortened. The growth rate may be determined by deriving various parameters from growth curves, such parameters may be: T-Mid (the time taken for plants to reach 50% of their maximal size) and T-90 (time taken for plants to reach 90% of their maximal size), amongst others.
According to a preferred feature of the present invention, performance of the methods of the invention gives plants having an increased growth rate relative to control plants. Therefore, according to the present invention, there is provided a method for increasing the growth rate of plants, which method comprises modulating expression in a plant of a nucleic acid encoding a PATL, or PRP38, or a GATA-like, or ADA2, or a WDR23-like polypeptide as defined herein.
An increase in yield and/or growth rate occurs under typical agricultural growth conditions which comprise the everyday stresses to which plants are exposed. Plants typically respond to exposure to stress by growing more slowly. In conditions of severe stress, the plant may even stop growing altogether. Mild or everyday stresses on the other hand are stresses to which a plant is exposed which does not result in the plant ceasing to grow altogether without the capacity to resume growth. Mild stress in the sense of the invention leads to a reduction in the growth of the stressed plants of less than 40%, 35% or 30%, preferably less than 25%, 20% or 15%, more preferably less than 14%, 13%, 12%, 11% or 10% or less in comparison to the control plant under non-stress conditions. Due to advances in agricultural practices (irrigation, fertilization, pesticide treatments) severe stresses are not often encountered in cultivated crop plants. A plant cultivated under typical agricultural conditions may often encounter mild stresses. As a consequence, the compromised growth induced by mild stress is often an undesirable feature for agriculture. Mild stresses are the everyday biotic and/or abiotic (environmental) stresses to which a plant is exposed. Abiotic stresses may be due to drought or excess water, anaerobic stress, salt stress, chemical toxicity, oxidative stress and hot, cold or freezing temperatures. The abiotic stress may be an osmotic stress caused by a water stress (particularly due to drought), salt stress, oxidative stress or an ionic stress.
Concerning the GATA-like, an increase in yield and/or growth rate occurs whether the plant is under non-stress conditions or whether the plant is exposed to various stresses compared to control plants. Concerning, WDR23, ncreased yield-related traits occur whether the plant is under non-stress conditions or whether the plant is exposed to various stresses compared to control plants grown under comparable conditions. Plants typically respond to exposure to stress by growing more slowly. In conditions of severe stress, the plant may even stop growing altogether. Mild stress on the other hand is defined herein as being any stress to which a plant is exposed which does not result in the plant ceasing to grow altogether without the capacity to resume growth. Mild stress in the sense of the invention leads to a reduction in the growth of the stressed plants of less than 40%, 35% or 30%, preferably less than 25%, 20% or 15%, more preferably less than 14%, 13%, 12%, 11% or 10% or less in comparison to the control plant under non-stress conditions. Due to advances in agricultural practices (irrigation, fertilization, pesticide treatments) severe stresses are not often encountered in cultivated crop plants. As a consequence, the compromised growth induced by mild stress is often an undesirable feature for agriculture. Mild stresses are the everyday biotic and/or abiotic (environmental) stresses to which a plant is exposed. Abiotic stresses may be due to drought or excess water, anaerobic stress, salt stress, chemical toxicity, oxidative stress and hot, cold or freezing temperatures. Biotic stresses are typically those stresses caused by pathogens, such as bacteria, viruses, fungi, nematodes, and insects. The term “non-stress” conditions as used herein are those environmental conditions that allow optimal growth of plants. Persons skilled in the art are aware of normal soil conditions and climatic conditions for a given location.
In particular, the methods of the present invention may be performed under non-stress conditions or under conditions of mild drought to give plants having increased yield relative to control plants. As reported in Wang et al. (Planta (2003) 218: 1-14), abiotic stress leads to a series of morphological, physiological, biochemical and molecular changes that adversely affect plant growth and productivity. Drought, salinity, extreme temperatures and oxidative stress are known to be interconnected and may induce growth and cellular damage through similar mechanisms. Rabbani et al. (Plant Physiol (2003) 133: 1755-1767) describes a particularly high degree of “cross talk” between drought stress and high-salinity stress. For example, drought and/or salinisation are manifested primarily as osmotic stress, resulting in the disruption of homeostasis and ion distribution in the cell. Oxidative stress, which frequently accompanies high or low temperature, salinity or drought stress, may cause denaturing of functional and structural proteins. As a consequence, these diverse environmental stresses often activate similar cell signalling pathways and cellular responses, such as the production of stress proteins, up-regulation of anti-oxidants, accumulation of compatible solutes and growth arrest. The term “non-stress” conditions are those environmental conditions that allow optimal growth of plants. Persons skilled in the art are aware of normal soil conditions and climatic conditions for a given location. The term non-stress conditions as used herein, encompasses the occasional or everyday mild stresses to which a plant is exposed, as defined herein, but does not encompass severe stresses.
Performance of the methods of the invention gives plants grown under non-stress conditions or under mild drought conditions increased yield relative to control plants grown under comparable conditions. Therefore, according to the present invention, there is provided a method for increasing yield in plants grown under non-stress conditions or under mild drought conditions, which method comprises modulating expression in a plant of a nucleic acid encoding a PATL, or PRP38, or a GATA-like, or ADA2, or a WDR23-like polypeptide.
Performance of the methods of the invention gives plants grown under conditions of nutrient deficiency, particularly under conditions of nitrogen deficiency, increased yield relative to control plants grown under comparable conditions. Therefore, according to the present invention, there is provided a method for increasing yield in plants grown under conditions of nutrient deficiency, which method comprises modulating expression in a plant of a nucleic acid encoding a PATL, or PRP38, or a GATA-like, or ADA2, or a WDR23-like polypeptide. Nutrient deficiency may result from a lack of nutrients such as nitrogen, phosphates and other phosphorous-containing compounds, potassium, calcium, cadmium, magnesium, manganese, iron and boron, amongst others. Concerning WDR23, preferably, reduced nutrient availablity is reduced nitrogen availability.
The present invention encompasses plants or parts thereof (including seeds) obtainable by the methods according to the present invention. The plants or parts thereof comprise a nucleic acid transgene encoding a PATL, or PRP38, or a GATA-like, or ADA2, or a WDR23-like polypeptide as defined above.
The invention also provides genetic constructs and vectors to facilitate introduction and/or expression in plants of nucleic acids encoding PATL, or PRP38, or a GATA-like, or ADA2, or a WDR23-like polypeptides. The gene constructs may be inserted into vectors, which may be commercially available, suitable for transforming into plants and suitable for expression of the gene of interest in the transformed cells. The invention also provides use of a gene construct as defined herein in the methods of the invention.
More specifically, the present invention provides a construct comprising:
-
- (a) a nucleic acid encoding a PATL, or PRP38, or a GATA-like, or ADA2, or a WDR23-like polypeptide as defined above;
- (b) one or more control sequences capable of driving expression of the nucleic acid sequence of (a); and optionally
- (c) a transcription termination sequence.
Preferably, the nucleic acid encoding a PATL, or PRP38, or a GATA-like, or ADA2, or a WDR23-like polypeptide is as defined above. The term “control sequence” and “termination sequence” are as defined herein.
Concerning WDR23, preferably, one of the control sequences of a construct is a constitutive promoter isolated from a plant genome. An example of a plant constitutive promoter is a GOS2 promoter, more preferably a rice GOS2 promoter, most preferably a GOS2 promoter as represented by SEQ ID NO: 272. Alternatively, one of the control sequences of a construct is a meristem-specific promoter isolated from a plant genome. An example of a plant meristem-specific promoter is a metallothionein (MT) promoter, more preferably a rice MT promoter, most preferably an MT promoter as represented by SEQ ID NO: 273.
Plants are transformed with a vector comprising any of the nucleic acids described above. The skilled artisan is well aware of the genetic elements that must be present on the vector in order to successfully transform, select and propagate host cells containing the sequence of interest. The sequence of interest is operably linked to one or more control sequences (at least to a promoter).
Advantageously, any type of promoter, whether natural or synthetic, may be used to increase expression of the nucleic acid sequence. A constitutive promoter is particularly useful in the methods. Preferably the constitutive promoter is also a ubiquitous promoter. Concerning the GATA-like polypeptides, the promoter is of medium strength See the “Definitions” section herein for definitions of the various promoter types.
It should be clear that the applicability of the present invention is not restricted to the PATL polypeptide-encoding nucleic acid represented by SEQ ID NO: 1, nor is the applicability of the invention restricted to expression of a PATL polypeptide-encoding nucleic acid when driven by a constitutive promoter, or when driven by a green-tissue promoter.
Furthermore it should be clear that the applicability of the invention is not restricted to the PRP38 polypeptide-encoding nucleic acid represented by SEQ ID NO: 76, nor is the applicability of the invention restricted to expression of a PRP38 polypeptide-encoding nucleic acid when driven by a constitutive promoter, or when driven by a root-specific promoter.
Also, it should be clear that the applicability of the present invention is not restricted to the GATA-like polypeptide-encoding nucleic acid represented by SEQ ID NO: 128, nor is the applicability of the invention restricted to expression of a GATA-like polypeptide-encoding nucleic acid when driven by a constitutive promoter.
Also, it should be clear that the applicability of the present invention is not restricted to the ADA2 polypeptide-encoding nucleic acid represented by SEQ ID NO: 181, nor is the applicability of the invention restricted to expression of an ADA2 polypeptide-encoding nucleic acid when driven by a constitutive promoter, or when driven by a green-tissue promoter.
Also, it should be clear that the applicability of the present invention is not restricted to a nucleic acid sequence encoding the WDR23-like polypeptide, as represented by SEQ ID NO: 215, nor is the applicability of the invention restricted to expression of a WDR23-like polypeptide-encoding nucleic acid sequence when driven by a constitutive or meristem-specific promoter.
The constitutive promoter is preferably a HMG (high mobility group), also called HMGP promoter, preferably a HMG promoter from rice. Further preferably the constitutive promoter is represented by a nucleic acid sequence substantially similar to SEQ ID NO: 33, most preferably the constitutive promoter is as represented by SEQ ID NO: 33. The green tissue specific promoter is preferably a EXP9 (Expansin), also called EXP, also called HMGP promoter, preferably a EXP promoter from rice. Further preferably the green tissue specific is represented by a nucleic acid sequence substantially similar to SEQ ID NO: 34, most preferably the constitutive promoter is as represented by SEQ ID NO: 34. See Table 2 in the “Definitions” section herein for reference describing HMG and EXP promoter and for further examples of constitutive and green tissue specific promoters.
Concerning PRP38, the constitutive promoter is preferably a GOS2 promoter, preferably a GOS2 promoter from rice. Further preferably the constitutive promoter is represented by a nucleic acid sequence substantially similar to SEQ ID NO: 127, most preferably the constitutive promoter is as represented by SEQ ID NO: 127. See Table ii in the “Definitions” section herein for further examples of constitutive promoters.
Concerning the GATA-like polypeptides, the constitutive promoter is preferably a GOS2 promoter, preferably a GOS2 promoter from rice. Further preferably the constitutive promoter is represented by a nucleic acid sequence substantially similar to SEQ ID NO: 135 most preferably the constitutive promoter is as represented by SEQ ID NO: 135. Optionally, one or more terminator sequences may be used in the construct introduced into a plant. Preferably, the construct comprises an expression cassette essentially similar or identical to SEQ ID NO 136, comprising the GOS2 promoter and the nucleic acid encoding the GATA-like polypeptide of SEQ ID NO: 129.
Concerning ADA2 polypeptides, the constitutive promoter is preferably a HMG (high mobility group), also called HMGP promoter, preferably a HMG promoter from rice. Further preferably the constitutive promoter is represented by a nucleic acid sequence substantially similar to SEQ ID NO: 213, most preferably the constitutive promoter is as represented by SEQ ID NO: 213. The green tissue specific promoter is preferably a EXP9 (Expansin), also called EXP, also called HMGP promoter, preferably a EXP promoter from rice. Further preferably the green tissue specific is represented by a nucleic acid sequence substantially similar to SEQ ID NO: 214, most preferably the constitutive promoter is as represented by SEQ ID NO: 214. See Table iii in the “Definitions” section herein for reference describing HMG and EXP promoter and for further examples of constitutive and green tissue specific promoters.
Additional regulatory elements may include transcriptional as well as translational enhancers. Those skilled in the art will be aware of terminator and enhancer sequences that may be suitable for use in performing the invention. An intron sequence may also be added to the 5′ untranslated region (UTR) or in the coding sequence to increase the amount of the mature message that accumulates in the cytosol, as described in the definitions section. Other control sequences (besides promoter, enhancer, silencer, intron sequences, 3′UTR and/or 5′UTR regions) may be protein and/or RNA stabilizing elements. Such sequences would be known or may readily be obtained by a person skilled in the art.
The genetic constructs of the invention may further include an origin of replication sequence that is required for maintenance and/or replication in a specific cell type. One example is when a genetic construct is required to be maintained in a bacterial cell as an episomal genetic element (e.g. plasmid or cosmid molecule). Preferred origins of replication include, but are not limited to, the f1-ori and colE1.
For the detection of the successful transfer of the nucleic acid sequences as used in the methods of the invention and/or selection of transgenic plants comprising these nucleic acids, it is advantageous to use marker genes (or reporter genes). Therefore, the genetic construct may optionally comprise a selectable marker gene. Selectable markers are described in more detail in the “definitions” section herein. The marker genes may be removed or excised from the transgenic cell once they are no longer needed. Techniques for marker removal are known in the art, useful techniques are described above in the definitions section.
The invention also provides a method for the production of transgenic plants having enhanced yield-related traits relative to control plants, comprising introduction and expression in a plant of any nucleic acid encoding a PATL, or PRP38, or a GATA-like, or ADA2, or a WDR23-like polypeptide as defined hereinabove.
More specifically, the present invention provides a method for the production of transgenic plants having increased enhanced yield-related traits, particularly increased seed yield, which method comprises:
- (i) introducing and expressing in a plant or plant cell a PATL, or PRP38, or ADA2 polypeptide-encoding nucleic acid; and
- (ii) cultivating the plant cell under conditions promoting plant growth and development, and optionally
- (iii) selecting for plants having enhanced yield-related traits
Concerning the GATA-like polypeptides, the present invention provides a method for the production of transgenic plants having increased enhanced yield-related traits, particularly increased TKW, which method comprises:
- (i) introducing and expressing in a plant or plant cell a GATA-like polypeptide, or a WDR23-like polypeptide-encoding nucleic acid; and
- (ii) cultivating the plant cell under conditions promoting plant growth and development.
The nucleic acid of (i) may be any of the nucleic acids capable of encoding a PATL, or PRP38, or a GATA-like, or ADA2, or a WDR23-like polypeptide as defined herein.
The nucleic acid may be introduced directly into a plant cell or into the plant itself (including introduction into a tissue, organ or any other part of a plant). According to a preferred feature of the present invention, the nucleic acid is preferably introduced into a plant by transformation. The term “transformation” is described in more detail in the “definitions” section herein.
The genetically modified plant cells can be regenerated via all methods with which the skilled worker is familiar. Suitable methods can be found in the abovementioned publications by S. D. Kung and R. Wu, Potrykus or Hofgen and Willmitzer.
Generally after transformation, plant cells or cell groupings are selected for the presence of one or more markers which are encoded by plant-expressible genes co-transferred with the gene of interest, following which the transformed material is regenerated into a whole plant. To select transformed plants, the plant material obtained in the transformation is, as a rule, subjected to selective conditions so that transformed plants can be distinguished from untransformed plants. For example, the seeds obtained in the above-described manner can be planted and, after an initial growing period, subjected to a suitable selection by spraying. A further possibility consists in growing the seeds, if appropriate after sterilization, on agar plates using a suitable selection agent so that only the transformed seeds can grow into plants. Alternatively, the transformed plants are screened for the presence of a selectable marker such as the ones described above.
Following DNA transfer and regeneration, putatively transformed plants may also be evaluated, for instance using Southern analysis, for the presence of the gene of interest, copy number and/or genomic organisation. Alternatively or additionally, expression levels of the newly introduced DNA may be monitored using Northern and/or Western analysis, both techniques being well known to persons having ordinary skill in the art.
The generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or T1) transformed plant may be selfed and homozygous second-generation (or T2) transformants selected, and the T2 plants may then further be propagated through classical breeding techniques. The generated transformed organisms may take a variety of forms. For example, they may be chimeras of transformed cells and non-transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed rootstock grafted to an untransformed scion).
The present invention clearly extends to any plant cell or plant produced by any of the methods described herein, and to all plant parts and propagules thereof. The present invention extends further to encompass the progeny of a primary transformed or transfected cell, tissue, organ or whole plant that has been produced by any of the aforementioned methods, the only requirement being that progeny exhibit the same genotypic and/or phenotypic characteristic(s) as those produced by the parent in the methods according to the invention.
The invention also includes host cells containing an isolated nucleic acid encoding a PATL, or PRP38, or a GATA-like, or ADA2, or a WDR23-like polypeptide as defined hereinabove. Preferred host cells according to the invention are plant cells. Host plants for the nucleic acids or the vector used in the method according to the invention, the expression cassette or construct or vector are, in principle, advantageously all plants, which are capable of synthesizing the polypeptides used in the inventive method.
The methods of the invention are advantageously applicable to any plant. Plants that are particularly useful in the methods of the invention include all plants which belong to the superfamily Viridiplantae, in particular monocotyledonous and dicotyledonous plants including fodder or forage legumes, ornamental plants, food crops, trees or shrubs. According to a preferred embodiment of the present invention, the plant is a crop plant. Examples of crop plants include soybean, sunflower, canola, alfalfa, rapeseed, cotton, tomato, potato and tobacco. Further preferably, the plant is a monocotyledonous plant. Examples of monocotyledonous plants include sugarcane. More preferably the plant is a cereal. Examples of cereals include rice, maize, wheat, barley, millet, rye, triticale, sorghum and oats.
The invention also extends to harvestable parts of a plant such as, but not limited to seeds, leaves, fruits, flowers, stems, roots, rhizomes, tubers and bulbs or also to an isolated nucleic acid sequence encoding a WDR23-like (as defined hereinabove) operably linked to a plant constitutive promoter. The invention furthermore relates to products derived, preferably directly derived, from a harvestable part of such a plant, such as dry pellets or powders, oil, fat and fatty acids, starch or proteins.
According to a preferred feature of the invention, the modulated expression is increased expression. Methods for increasing expression of nucleic acids or genes, or gene products, are well documented in the art and examples are provided in the definitions section.
As mentioned above, a preferred method for modulating expression of a nucleic acid encoding a PATL, or PRP38, or a GATA-like, or ADA2, or a WDR23-like polypeptide is by introducing and expressing in a plant a nucleic acid encoding a PATL, or PRP38, or a GATA-like, or ADA2, or a WDR23-like polypeptide; however the effects of performing the method, i.e. enhancing yield-related traits may also be achieved using other well known techniques, including but not limited to T-DNA activation tagging, TILLING, homologous recombination. A description of these techniques is provided in the definitions section.
The present invention also encompasses use of nucleic acids encoding PATL, or PRP38, or a GATA-like, or ADA2, or a WDR23-like polypeptides as described herein and use of these PATL, or PRP38, or a GATA-like, or ADA2, or a WDR23-like polypeptides in enhancing any of the aforementioned yield-related traits in plants, under normal growth conditions, under abiotic stress growth (preferably osmotic stress growth conditions) conditions, and under growth conditions of reduced nutrient availability, preferably under conditions of reduced nitrogen availability.
Nucleic acids encoding PATL, or PRP38, or a GATA-like, or ADA2, or a WDR23-like polypeptide described herein, or the PATL, or PRP38, or a GATA-like, or ADA2, or a WDR23-like polypeptides themselves, may find use in breeding programmes in which a DNA marker is identified which may be genetically linked to a PATL, or PRP38, or a GATA-like, or ADA2, or a WDR23-like polypeptide-encoding gene. The nucleic acids/genes, or the PATL, or PRP38, or a GATA-like, or ADA2, or a WDR23-like polypeptides themselves may be used to define a molecular marker. This DNA or protein marker may then be used in breeding programmes to select plants having enhanced yield-related traits as defined hereinabove in the methods of the invention.
Allelic variants of a PATL, or PRP38, or a GATA-like, or ADA2, or a WDR23-like polypeptide-encoding nucleic acid/gene may also find use in marker-assisted breeding programmes. Such breeding programmes sometimes require introduction of allelic variation by mutagenic treatment of the plants, using for example EMS mutagenesis; alternatively, the programme may start with a collection of allelic variants of so called “natural” origin caused unintentionally. Identification of allelic variants then takes place, for example, by PCR. This is followed by a step for selection of superior allelic variants of the sequence in question and which give increased yield. Selection is typically carried out by monitoring growth performance of plants containing different allelic variants of the sequence in question. Growth performance may be monitored in a greenhouse or in the field. Further optional steps include crossing plants in which the superior allelic variant was identified with another plant. This could be used, for example, to make a combination of interesting phenotypic features.
Nucleic acids encoding PATL, or PRP38, or a GATA-like, or ADA2, or a WDR23-like polypeptides may also be used as probes for genetically and physically mapping the genes that they are a part of, and as markers for traits linked to those genes. Such information may be useful in plant breeding in order to develop lines with desired phenotypes. Such use of PATL, or PRP38, or a GATA-like, or ADA2, or a WDR23-like polypeptide-encoding nucleic acids requires only a nucleic acid sequence of at least 15 nucleotides in length. The PATL, or PRP38, or a GATA-like, or ADA2, or a WDR23-like polypeptide-encoding nucleic acids may be used as restriction fragment length polymorphism (RFLP) markers. Southern blots (Sambrook J, Fritsch E F and Maniatis T (1989) Molecular Cloning, A Laboratory Manual) of restriction-digested plant genomic DNA may be probed with the PATL, or PRP38, or GATA-like, or ADA2, or a WDR23-like-encoding nucleic acids. The resulting banding patterns may then be subjected to genetic analyses using computer programs such as MapMaker (Lander et al. (1987) Genomics 1: 174-181) in order to construct a genetic map. In addition, the nucleic acids may be used to probe Southern blots containing restriction endonuclease-treated genomic DNAs of a set of individuals representing parent and progeny of a defined genetic cross. Segregation of the DNA polymorphisms is noted and used to calculate the position of the PATL, or PRP38, or a GATA-like, or ADA2, or a WDR23-like polypeptide-encoding nucleic acid in the genetic map previously obtained using this population (Botstein et al. (1980) Am. J. Hum. Genet. 32:314-331).
The production and use of plant gene-derived probes for use in genetic mapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol. Reporter 4: 37-41. Numerous publications describe genetic mapping of specific cDNA clones using the methodology outlined above or variations thereof. For example, F2 intercross populations, backcross populations, randomly mated populations, near isogenic lines, and other sets of individuals may be used for mapping. Such methodologies are well known to those skilled in the art.
The nucleic acid probes may also be used for physical mapping (i.e., placement of sequences on physical maps; see Hoheisel et al. In: Non-mammalian Genomic Analysis: A Practical Guide, Academic press 1996, pp. 319-346, and references cited therein).
In another embodiment, the nucleic acid probes may be used in direct fluorescence in situ hybridisation (FISH) mapping (Trask (1991) Trends Genet. 7:149-154). Although current methods of FISH mapping favour use of large clones (several kb to several hundred kb; see Laan et al. (1995) Genome Res. 5:13-20), improvements in sensitivity may allow performance of FISH mapping using shorter probes.
A variety of nucleic acid amplification-based methods for genetic and physical mapping may be carried out using the nucleic acids. Examples include allele-specific amplification (Kazazian (1989) J. Lab. Clin. Med 11:95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332), allele-specific ligation (Landegren et al. (1988) Science 241:1077-1080), nucleotide extension reactions (Sokolov (1990) Nucleic Acid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997) Nat. Genet. 7:22-28) and Happy Mapping (Dear and Cook (1989) Nucleic Acid Res. 17:6795-6807). For these methods, the sequence of a nucleic acid is used to design and produce primer pairs for use in the amplification reaction or in primer extension reactions. The design of such primers is well known to those skilled in the art. In methods employing FOR-based genetic mapping, it may be necessary to identify DNA sequence differences between the parents of the mapping cross in the region corresponding to the instant nucleic acid sequence. This, however, is generally not necessary for mapping methods.
The methods according to the present invention result in plants having enhanced yield-related traits, as described hereinbefore. These traits may also be combined with other economically advantageous traits, such as further yield-enhancing traits, tolerance to other abiotic and biotic stresses, traits modifying various architectural features and/or biochemical and/or physiological features.
In one embodiment the invention relates to subject mater summarized as follows:
Item 1A A method for enhancing yield-related traits in plants relative to control plants, comprising modulating expression in a plant of a nucleic acid encoding a PATELLIN polypeptide and optionally selecting for plants having enhanced yield-related traits.
Item 2A Method according to item 1A, wherein said PATELLIN polypeptide comprises at least one of the following domains:
(i) A SEC14 domain as represented by SEQ ID NO: 71: Ipeldsvvfyrgadreghpvcynvygefqdkdlyekafgdeekrerflkwriqllergilsqldfspsgicsmvqvtdlknsppmlgkhr avtrgavallqdnypefiakkvfinvpwwylaankmmspfltqrtkskfifaspaksaetlfryiapeqvpvqfgglfk or a domain having in increasing order of preference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82%, 85%, 90%, 92%, 95%, 97% or more sequence identity to the domain represented by SEQ ID NO: 71 or to any SEC14 domain as present in any of the polypeptides of Table A;
(ii) A GOLD domain as represented by SEQ ID NO: 72: sdavteltikpssketveipvtenstigwelrvlgwevsygaeftpdaeggytvivqktrkvpaneepimkgsfkvgepgkivltinnp askkkkllyrskv, or a domain having in increasing order of preference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82%, 85%, 90%, 92%, 95%, 97% or more sequence identity to the domain represented by SEQ ID NO: 72 or to any GOLD domain as present in any of the polypeptides of Table A.
Item 3A Method according to item 1A or 2A, wherein said modulated expression is effected by introducing and expressing in a plant a nucleic acid encoding a PATELLIN polypeptide.
Item 4A Method according to any preceding item, wherein said nucleic acid encoding a PATELLIN polypeptide encodes any one of the proteins listed in Table A or is a portion of such a nucleic acid, or a nucleic acid capable of hybridising with such a nucleic acid.
Item 5A Method according to any preceding item, wherein said nucleic acid sequence encodes an orthologue or paralogue of any of the proteins given in Table A.
Item 6A Method according to any preceding item, wherein said enhanced yield-related traits comprise increased yield, preferably seed yield relative to control plants.
Item 7A Method according to any one of items 1A to 6A, wherein said enhanced yield-related traits are obtained under conditions of nitrogen deficiency.
Item 8A Method according to any one of items 3A to 7A, wherein said nucleic acid is operably linked to a constitutive promoter, preferably to a GOS2 promoter, most preferably to a GOS2 promoter from rice.
Item 9A Method according to any preceding item, wherein said nucleic acid encoding a PATELLIN polypeptide is of plant origin, preferably from a dicotyledonous plant, further preferably from the family Poaceae, more preferably from the genus Oryza, most preferably from Oryza sativa.
Item 10A Plant or part thereof, including seeds, obtainable by a method according to any preceding item, wherein said plant or part thereof comprises a recombinant nucleic acid encoding a PATELLIN polypeptide.
Item 11A An isolated nucleic acid molecule comprising any one of the following features:
(i) a nucleic acid represented by SEQ ID NO: 9; SEQ ID NO: 11; SEQ ID NO: 13; SEQ ID NO: 15; SEQ ID NO: 17; SEQ ID NO: 19; SEQ ID NO: 21; SEQ ID NO: 23; SEQ ID NO: 25 and SEQ ID NO: 27;
(ii) a nucleic acid fragment that is complementary to any one of the SEQ ID NOs given in (i);
(iii) a nucleic acid encoding a PATELLIN polypeptide having, in increasing order of preference, at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of the amino acid sequences given in SEQ ID NO: 10; SEQ ID NO: 12; SEQ ID NO: 14; SEQ ID NO: 16; SEQ ID NO: 18; SEQ ID NO: 20; SEQ ID NO: 22; SEQ ID NO: 24; SEQ ID NO: 26 and SEQ ID NO: 28;
(iv) a nucleic acid capable of hybridizing under stringent conditions to any one of the nucleic acids given in (i), (ii) or (iii) above.
Item 12A An isolated polypeptide comprising:
(I) an amino acid sequence having, in increasing order of preference, at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of the amino acid sequences given in SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24; SEQ ID NO: 26 and SEQ ID NO: 28;
(ii) derivatives of any of the amino acid sequences given in (i).
Item 13A Construct comprising:
(I) nucleic acid encoding a PATELLIN polypeptide as defined in items 1A, 2A or 12A, or a nucleic acid according to item 11;
(ii) one or more control sequences capable of driving expression of the nucleic acid sequence of (a); and optionally
(iii) a transcription termination sequence.
Item 14A Construct according to item 13A, wherein one of said control sequences is a constitutive promoter, preferably a GOS2 promoter, most preferably a GOS2 promoter from rice.
Item 15A Use of a construct according to item 13A or 14A in a method for making plants having increased yield, particularly increased biomass and/or increased seed yield relative to control plants.
Item 16A Plant, plant part or plant cell transformed with a construct according to item 13A or 14A.
Item 17A Method for the production of a transgenic plant having increased yield, preferably increased seed yield relative to control plants, comprising:
(i) introducing and expressing in a plant a nucleic acid encoding a PATELLIN polypeptide as defined in item 1A, 2A or 12A, or a nucleic acid according to item 11A; and
(ii) cultivating the plant cell under conditions promoting plant growth and development; and optionally
(iii) selecting for plants having enhanced yield-related traits
Item 18A Transgenic plant having increased yield, particularly increased seed yield, relative to control plants, resulting from modulated expression of a nucleic acid encoding a PATELLIN polypeptide as defined in item 1A or 2A, or a transgenic plant cell derived from said transgenic plant.
Item 19A Transgenic plant according to item 11A, 16A or 18A, or a transgenic plant cell derived thereof, wherein said plant is a crop plant or a monocot or a cereal, such as rice, maize, wheat, barley, millet, rye, triticale, sorghum and oats.
Item 20A Harvestable parts of a plant according to item 19A, wherein said harvestable parts are preferably shoot biomass and/or seeds.
Item 21A Products derived from a plant according to item 19A and/or from harvestable parts of a plant according to item 20A.
Item 22A Use of a nucleic acid encoding a PATELLIN polypeptide in increasing yield, particularly in increasing seed yield and/or shoot biomass in plants, relative to control plants.
Item 1B A method for enhancing yield-related traits in plants relative to control plants, comprising modulating expression in a plant of a nucleic acid encoding a PRP38 polypeptide.
Item 2B Method according to item 1B, wherein said PRP38 polypeptide comprises one or more of the following motifs:
(i) A DUF1777 domain
(ii) An RS domain
(iii) Motif 1a (SEQ ID NO: 120) wherein any amino acid residue may be substituted by a conservative amino acid and/or up to 50% of the amino acid residues may be substituted by a non conservative amino acid.
(iv) Motif 1a (SEQ ID NO: 121) wherein any amino acid residue may be substituted by a conservative amino acid and/or up to 50% of the amino acid residues may be substituted by a non conservative amino acid.
(v) Motif 1a (SEQ ID NO: 122) wherein any amino acid residue may be substituted by a conservative amino acid and/or up to 50% of the amino acid residues may be substituted by a non conservative amino acid.
Item 3B Method according to item 1B or 2B, wherein said modulated expression is effected by introducing and expressing in a plant a nucleic acid encoding a PRP38 polypeptide.
Item 4B Method according to any preceding item, wherein said nucleic acid encoding a PRP38 polypeptide encodes any one of the proteins listed in Table A or is a portion of such a nucleic acid, or a nucleic acid capable of hybridising with such a nucleic acid.
Item 5B Method according to any preceding item, wherein said nucleic acid sequence encodes an orthologue or paralogue of any of the proteins given in Table A.
Item 6B Method according to any preceding item, wherein said enhanced yield-related traits comprise increased yield, preferably increased biomass and/or increased seed yield relative to control plants.
Item 7B Method according to any one of items 1B to 6B, wherein said enhanced yield-related traits are obtained under non-stress conditions.
Item 8B Method according to any one of items 1B to 6B, wherein said enhanced yield-related traits are obtained under conditions of nitrogen deficiency.
Item 9B Method according to any one of items 3B to 8B, wherein said nucleic acid is operably linked to a constitutive promoter, preferably to a GOS2 promoter, most preferably to a GOS2 promoter from rice.
Item 10B Method according to any preceding item, wherein said nucleic acid encoding a PRP38 polypeptide is of plant origin, preferably from a dicotyledonous plant, further preferably from the family Brassicaceae, more preferably from the genus Arabidopsis, most preferably from Arabidopsis thaliana.
Item 11B Plant or part thereof, including seeds, obtainable by a method according to any preceding item, wherein said plant or part thereof comprises a recombinant nucleic acid encoding a PRP38 polypeptide.
Item 12B Construct comprising:
(i) nucleic acid encoding a PRP38 polypeptide as defined in items 1 or 2;
(ii) one or more control sequences capable of driving expression of the nucleic acid sequence of (a); and optionally
(iii) a transcription termination sequence.
Item 13B Construct according to item 12,B wherein one of said control sequences is a constitutive promoter, preferably a GOS2 promoter, most preferably a GOS2 promoter from rice.
Item 14B Use of a construct according to item 12B or 13B in a method for making plants having increased yield, particularly increased biomass and/or increased seed yield relative to control plants.
Item 15B Plant, plant part or plant cell transformed with a construct according to item 12B or 13B.
Item 16B Method for the production of a transgenic plant having increased yield, particularly increased biomass and/or increased seed yield relative to control plants, comprising:
(i) introducing and expressing in a plant a nucleic acid encoding a PRP38 polypeptide as defined in item 1B or 2B; and
(ii) cultivating the plant cell under conditions promoting plant growth and development.
Item 17B Transgenic plant having increased yield, particularly increased biomass and/or increased seed yield, relative to control plants, resulting from modulated expression of a nucleic acid encoding a PRP38 polypeptide as defined in item 1B or 2B, or a transgenic plant cell derived from said transgenic plant.
Item 18B Transgenic plant according to item 11B, 15B or 17B, or a transgenic plant cell derived thereof, wherein said plant is a crop plant or a monocot or a cereal, such as rice, maize, wheat, barley, millet, rye, triticale, sorghum and oats.
Item 19B Harvestable parts of a plant according to item 18B, wherein said harvestable parts are preferably shoot biomass and/or seeds.
Item 20B Products derived from a plant according to item 18B and/or from harvestable parts of a plant according to item 19B.
Item 21B Use of a nucleic acid encoding a PRP38 polypeptide in increasing yield, particularly in increasing seed yield and/or shoot biomass in plants, relative to control plants.
Item 1C A method for increasing one or more of Thousand Kernel Weight, total weight of seeds and number of filled seeds in plants relative to control plants, comprising modulating expression in a plant of a nucleic acid encoding a GATA-like polypeptide, wherein said GATA-like polypeptide belongs to subfamily II of GATA transcription factors and comprises a GATA domain.
Item 2C Method according to item 1C, wherein said GATA-like polypeptide comprises one or more of the following motifs:
Item 3C Method according to item 1C or 2C, wherein said modulated expression is effected by introducing and expressing in a plant a nucleic acid encoding a GATA-like polypeptide.
Item 4C Method according to any preceding item, wherein said nucleic acid encoding a GATA-like polypeptide encodes any one of the proteins listed in Table A or is a portion of such a nucleic acid, or a nucleic acid capable of hybridising with such a nucleic acid.
Item 5C Method according to any preceding item, wherein said nucleic acid sequence encodes an orthologue or paralogue of any of the proteins given in Table A.
Item 6C Method according to any one of items 1C to 5C, wherein said enhanced yield-related traits are obtained under non-stress conditions.
Item 7C Method according to any one of items 3C to 6C, wherein said nucleic acid is operably linked to a constitutive promoter, preferably to a GOS2 promoter, most preferably to a GOS2 promoter from rice.
Item 8C Method according to any preceding item, wherein said nucleic acid encoding a GATA-like polypeptide is of plant origin, preferably from a monocotyledonous plant, further preferably from the family Poaceae, more preferably from the genus Oryza, most preferably from Oryza sativa.
Item 9C Plant or part thereof, including seeds, obtainable by a method according to any preceding item, wherein said plant or part thereof comprises a recombinant nucleic acid encoding a GATA-like polypeptide operably linked to constitutive promoter of plant origin, preferably a GOS2 promoter, more preferably a GOS2 promoter from rice.
Item 10C Construct comprising:
(i) nucleic acid encoding a GATA-like polypeptide as defined in items 1C or 2C;
(ii) one or more control sequences of plant origin capable of driving expression of the nucleic acid sequence of (a); and optionally
(iii) a transcription termination sequence.
Item 110 Construct according to item 100, wherein one of said control sequences is a constitutive promoter, preferably a GOS2 promoter, most preferably a GOS2 promoter from rice.
Item 12C Use of a construct according to item 100 or 110 in a method for making plants having increased seed yield, comprising one or more of Thousand Kernel Weight, total weight of seeds and number of filled seeds, relative to control plants.
Item 13C Plant, plant part or plant cell transformed with a construct according to item 100 or 110.
Item 14C Method for the production of a transgenic plant having increased yield, particularly increased biomass and/or increased seed yield relative to control plants, comprising:
(i) introducing and expressing in a plant a nucleic acid encoding a GATA-like polypeptide as defined in item 1C or 2C and operably linked to a constitutive promoter of plant origin; and
(ii) cultivating the plant cell under conditions promoting plant growth and development.
Item 15C Transgenic plant having increased Thousand Kernel Weight, relative to control plants, resulting from modulated expression of a nucleic acid encoding a GATA-like polypeptide as defined in item 1C or 2C, or a transgenic plant cell derived from said transgenic plant.
Item 16C Transgenic plant according to item 9C, 13C or 15C, or a transgenic plant cell derived thereof, wherein said plant is a crop plant or a monocot or a cereal, such as rice, maize, wheat, barley, millet, rye, triticale, sorghum and oats.
Item 17C Harvestable parts of a plant according to item 17C, wherein said harvestable parts are preferably seeds.
Item 18C Products derived from a plant according to item 16C and/or from harvestable parts of a plant according to item 17C.
Item 19C Use of a nucleic acid encoding a GATA-like polypeptide in increasing one or more of Thousand Kernel Weight, total weight of seeds and number of filled seeds in plants, relative to control plants.
Item 1D A method for enhancing yield-related traits in plants relative to control plants, comprising modulating expression in a plant of a nucleic acid encoding an ADA2 polypeptide and optionally selecting for plants having enhanced yield-related traits.
Item 2D Method according to item 1D, wherein said ADA2 polypeptide comprises two or more of the following motifs:
(i) A Zinc (Zn) finger ZZ type domain as represented by SEQ ID NO: 207: kpglyccnycdkdlsglvrfkcavcmdfdlcvecfsvgvelnrhkn or a domain having in increasing order of preference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82%, 85%, 90%, 92%, 95%, 97% or more sequence identity to the domain represented by SEQ ID NO: 207 or to any Zn finger ZZ type domain as present in any one of the polypeptides of Table A;
(ii) A SANT DNA binding domain as represented by SEQ ID NO: 208: vtsdwnadeeillleaiatygfgnwkevadhvgsktttecikhfnsaym, or a domain having in increasing order of preference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82%, 85%, 90%, 92%, 95%, 97% or more sequence identity to the domain represented by SEQ ID NO: 208 or to any SANT domain as present in any one of the polypeptides of Table A;
(iii) A Ca binding EF hand domain as represented by SEQ ID NO: 209: dndaeqlladmef, or a domain having in increasing order of preference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82%, 85%, 90%, 92%, 95%, 97% or more sequence identity to the domain represented by SEQ ID NO: 209 or to any Ca binding EF hand domain as present in any one of the polypeptides of Table A;
(iv) A SWIRM domain as represented by SEQ ID NO: 210: priysgldtwdydgllgadllsetekkmcnetrilpyhylkmldiltreikkgqikkksdaysffkvepskydryydmlyhkgigdst, or a domain having in increasing order of preference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82%, 85%, 90%, 92%, 95%, 97% or more sequence identity to the domain represented by SEQ ID NO: 210 or to any SWRIM domain as present in any one of the polypeptides of Table A;
Item 3D Method according to item 1D or 2D, wherein said modulated expression is effected by introducing and expressing in a plant a nucleic acid encoding an ADA2 polypeptide.
Item 4D Method according to any preceding item, wherein said nucleic acid encoding an ADA2 polypeptide encodes any one of the proteins listed in Table A or is a portion of such a nucleic acid, or a nucleic acid capable of hybridising with such a nucleic acid.
Item 5D Method according to any preceding item, wherein said nucleic acid sequence encodes an orthologue or paralogue of any of the proteins given in Table A.
Item 6D Method according to any preceding item, wherein said enhanced yield-related traits comprise increased yield, preferably seed yield relative to control plants.
Item 7D Method according to any one of items 1D to 6D, wherein said enhanced yield-related traits are obtained under mild-stress conditions.
Item 8D Method according to any one of items 1D to 6D, wherein said enhanced yield-related traits are obtained under conditions of nitrogen deficiency.
Item 9D Method according to any one of items 3D to 8D, wherein said nucleic acid is operably linked to a constitutive promoter, preferably to a HMGP promoter, most preferably to a HMGP promoter from rice.
Item 10D Method according to any preceding item, wherein said nucleic acid encoding an ADA2 polypeptide is of plant origin, preferably from a dicotyledonous plant, further preferably from the family Brassicaceae, more preferably from the genus Arabidopsis, most preferably from Arabidopsis thaliana.
Item 11 D Plant or part thereof, including seeds, obtainable by a method according to any preceding item, wherein said plant or part thereof comprises a recombinant nucleic acid encoding an ADA2 polypeptide.
Item 12D Construct comprising:
(i) nucleic acid encoding an ADA2 polypeptide as defined in items 1D or 2D;
(ii) one or more control sequences capable of driving expression of the nucleic acid sequence of (a); and optionally
(iii) a transcription termination sequence.
Item 13D Construct according to item 12D, wherein one of said control sequences is a constitutive promoter, preferably a HMGP promoter, most preferably a HMGP promoter from rice.
Item 14D Use of a construct according to item 12D or 13D in a method for making plants having increased yield, particularly increased biomass and/or increased seed yield relative to control plants.
Item 15D Plant, plant part or plant cell transformed with a construct according to item 12D or 13D.
Item 16D Method for the production of a transgenic plant having increased yield, particularly increased seed yield relative to control plants, comprising:
(i) introducing and expressing in a plant a nucleic acid encoding an ADA2 polypeptide as defined in item 1D or 2D; and
(ii) cultivating the plant cell under conditions promoting plant growth and development; and optionally
(iii) selecting for plants having enhanced yield-related traits
Item 17D Transgenic plant having increased yield, particularly increased seed yield, relative to control plants, resulting from modulated expression of a nucleic acid encoding an ADA2 polypeptide as defined in item 1D or 2D, or a transgenic plant cell derived from said transgenic plant.
Item 18D Transgenic plant according to item 11D, 15D or 17D, or a transgenic plant cell derived thereof, wherein said plant is a crop plant or a monocot or a cereal, such as rice, maize, wheat, barley, millet, rye, triticale, sorghum and oats.
Item 19D Harvestable parts of a plant according to item 19, wherein said harvestable parts are preferably shoot biomass and/or seeds.
Item 20D Products derived from a plant according to item 18D and/or from harvestable parts of a plant according to item 19D.
Item 21 D Use of a nucleic acid encoding an ADA2 polypeptide in increasing yield, particularly in increasing seed yield and/or shoot biomass in plants, relative to control plants.
Item 1E An isolated nucleic acid sequence encoding a WD40 repeat (WDR) 23-like polypeptide comprising:
(i) an isolated nucleic acid sequence as represented by SEQ ID NO: 219, SEQ ID NO: 225, or SEQ ID NO: 229;
(ii) the complement of an isolated nucleic acid sequence as represented by SEQ ID NO: 219, SEQ ID NO: 225, or SEQ ID NO: 229;
(iii) an isolated nucleic acid sequence encoding a polypeptide sequence as represented by SEQ ID NO: 220, SEQ ID NO: 226, or SEQ ID NO: 230;
(iv) an isolated nucleic acid sequence deduced from a polypeptide sequence as represented by SEQ ID NO: 220, SEQ ID NO: 226, or SEQ ID NO: 230, as a result of the degeneracy of the genetic code;
(v) an isolated nucleic acid sequence capable of stringently hybridising to a nucleic acid sequence as represented by SEQ ID NO: 219, SEQ ID NO: 225, or SEQ ID NO: 229, or to its complement;
(vi) an isolated nucleic acid sequence which encodes a polypeptide having in increasing order of preference at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity with a polypeptide sequence as represented by SEQ ID NO: 220, SEQ ID NO: 226, or SEQ ID NO: 230;
(vii) an isolated nucleic acid sequence which encodes a polypeptide comprising a domain having, in increasing order of preference, at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity with a Conserved Domain as represented by SEQ ID NO: 271.
Item 2E An isolated WDR23-like polypeptide comprising:
(i) a polypeptide sequence as represented by SEQ ID NO: 220, SEQ ID NO: 226, or SEQ ID NO: 230;
(ii) a polypeptide sequence having, in increasing order of preference, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% amino acid sequence identity with a polypeptide sequence as represented by SEQ ID NO: 220, SEQ ID NO: 226, or SEQ ID NO: 230;
(I) a polypeptide comprising a domain having, in increasing order of preference, at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity with a Conserved Domain as represented by SEQ ID NO: 271;
(ii) derivatives of any of the polypeptide sequences given in (i) to (iii) above.
Item 3E A method for increasing yield-related traits in plants relative to control plants, comprising increasing expression in a plant of a nucleic acid sequence encoding a WDR23-like polypeptide, which WDR23-like polypeptide comprises a domain having at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid sequence identity to a Conserved Domain (CD) as represented by SEQ ID NO: 271, and optionally selecting for plants having increased yield-related traits.
Item 4E Method according to item 3E, wherein said WDR23-like polypeptide comprises:
(i) at least four WD40 repeats with a PFAM accession PF00400; and (ii) at least two conserved DxR motifs at the end of two consecutive WD40 repeats.
Item 5E Method according to item 3E or 4E, wherein said WDR23-like polypeptide has, in increasing order of preference, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid sequence identity to the WDR23-like polypeptide as represented by SEQ ID NO: 216, or to any of the polypeptide sequences given in Table A herein, or to a WDR23-like polypeptide as defined in item 2E.
Item 6E Method according to any one of items 3E to 5E, wherein said nucleic acid sequence encoding a WDR23-like polypeptide is represented by any one of the nucleic acid sequence SEQ ID NOs given in Table A or a portion thereof, or a sequence capable of hybridising with any one of the nucleic acid sequences SEQ ID NOs given in Table A.
Item 7E Method according to any one of imtes 3E to 6E, wherein said nucleic acid sequence encodes an orthologue or paralogue of any of the polypeptide sequence SEQ ID NOs given in Table A.
Item 8E Method according to any one of items 3E to 7E, wherein said increased expression is effected by any one or more of: T-DNA activation tagging, TILLING, or homologous recombination.
Item 9E Method according to any one of items 3E to 8E, wherein said increased expression is effected by introducing and expressing in a plant a nucleic acid sequence encoding a WDR23-like polypeptide.
Item 10E Method according to any one of items 3E to 9E, wherein said increased yield-related trait is one or more of: increased total seed yield per plant, increased seed filling rate, increased number of filled seeds, increased harvest index, or increased thousand kernel weight.
Item 11E Method according to any one of items 3E to 10E, wherein said nucleic acid sequence is operably linked to a constitutive promoter, preferably to a plant constitutive promoter, more preferably to a GOS2 promoter, most preferably to a GOS2 promoter from rice as represented by SEQ ID NO: 272.
Item 12E Method according to any one of items 3E to 10E, wherein said nucleic acid sequence is operably linked to a meristem-specific promoter, preferably to a plant metallothionein promoter, more preferably to a metallothionein promoter from rice as represented by SEQ ID NO: 273.
Item 13E Method according to any one of items 3E to 12E, wherein said nucleic acid sequence encoding a WDR23-like polypeptide is from the plant kingdom, preferably from a dicotyledonous plant, more preferably from the Brassicaceae family, most preferably from Arabidopsis thaliana.
Item 14E Plants, parts thereof (including seeds), or plant cells obtainable by a method according to any one of items 3E to 13E, wherein said plant, part or cell thereof comprises an isolated nucleic acid transgene encoding a WDR23-like polypeptide operably linked to a plant constitutive promoter.
Item 15E Plants, parts thereof (including seeds), or plant cells comprising an isolated nucleic acid transgene according to item 1E, or comprising an isolated nucleic acid sequence encoding a WDR23-like polypeptide according to item 2E.
Item 16E Construct comprising:
(a) a nucleic acid sequence encoding a WDR23-like polypeptide as defined in any one of items 1E, 2E, or 3E to 7E;
(b) one or more control sequences capable of driving expression of the nucleic acid sequence of (a); and optionally
(c) transcription termination sequence.
Item 17E Construct according to item 16E, wherein said control sequence is a plant constitutive promoter, preferably a GOS2 promoter, more preferably a rice GOS2 promoter, most preferably a GOS2 promoter as represented by SEQ ID NO: 272.
Item 18E Construct according to item 16E, wherein said control sequence is a meristem-specific promoter, preferably a metallothionein (MT) promoter, more preferably a rice MT promoter, most preferably an MT promoter as represented by SEQ ID NO: 273.
Item 19E Use of a construct according any one of items 16E to18E in a method for making plants having increased yield-related traits relative to control plants, which increased yield-related traits are one or more of: increased total seed yield per plant, increased seed filling rate, increased number of filled seeds, increased harvest index, or increased thousand kernel weight.
Item 20E Plant, plant part or plant cell transformed with a construct according to any one of items 16E to18E.
Item 21 E Method for the production of transgenic plants having increased yield-related traits relative to control plants, comprising:
(i) introducing and expressing in a plant, plant part, or plant cell, a nucleic acid sequence encoding a WDR23-like polypeptide as defined in any one of items 1E or 3E to 7E; and
(ii) cultivating the plant cell, plant part, or plant under conditions promoting plant growth and development.
Item 22E Transgenic plant having increased yield-related traits relative to control plants, resulting from increased expression of a nucleic acid sequence encoding a WDR23-like polypeptide as defined in any one of items 1E, 3E to 7E, operably linked to a plant expressible promoter, or a transgenic plant cell or transgenic plant part derived from said transgenic plant.
Item 23E Transgenic plant according to item 14E, 15E, 20E or 22E, wherein said plant is a crop plant or a monocot or a cereal, such as rice, maize, wheat, barley, millet, rye, triticale, sorghum and oats, or a transgenic plant cell derived from said transgenic plant.
Item 24E Harvestable parts of a plant according to item 23E, comprising an isolated nucleic acid sequence encoding a WDR23-like polypeptide, wherein said harvestable parts are preferably seeds.
Item 25E Products derived from a plant according to item 23E and/or from harvestable parts of a plant according to item 24E.
Item 26E Use of a nucleic acid sequence encoding a WDR23-like polypeptide as defined in any one of items 1E, 3E to 7E in increasing yield-related traits, comprising one or more of: increased total seed yield per plant, increased seed filling rate, increased number of filled seeds, increased harvest index, or increased thousand kernel weight.
The present invention will now be described with reference to the following figures in which:
BLADE i: Glyma_WDR23 LIKE (SEQ ID NO: 222); Pruar_WDR23 LIKE (SEQ ID NO: 244); Horvu_WDR23 LIKE II (SEQ ID NO: 228); Triae_WDR23 LIKE II (SEQ ID NO: 250); Sacof_WDR23 LIKE II (SEQ ID NO: 246); Zeama_WDR23 LIKE II (SEQ ID NO: 256); Orysa_WDR23 LIKE (SEQ ID NO: 238); Triae_WDR23 LIKE (SEQ ID NO: 248); Zeama_WDR23 LIKE (SEQ ID NO: 254); Helan_WDR23 LIKE (SEQ ID NO: 226); Lyces_WDR23 LIKE (SEQ ID NO: 234); Liter_WDR23 LIKE (SEQ ID NO: 232); Glyma_WDR23 LIKE II (SEQ ID NO: 260); Medtr_WDR23 LIKE (SEQ ID NO: 236); Arath_WDR23 LIKE (SEQ ID NO: 216); Linus_WDR23 LIKE (SEQ ID NO: 230); Vitis_WDR23 LIKE (SEQ ID NO: 252); Pinra_WDR23 LIKE (SEQ ID NO: 240); Aqufo_WDR23 LIKE (SEQ ID NO: 218); Poptr_WDR23 LIKE (SEQ ID NO: 242); Homsa_WDR23 (SEQ ID NO: 280); Aspn_LEC14B (SEQ ID NO: 282); and Consensus (SEQ ID NO: 284). BLADE i+1: Glyma_WDR23 LIKE (SEQ ID NO: 222); Pruar_WDR23 LIKE (SEQ ID NO: 244); Horvu_WDR23 LIKE II (SEQ ID NO: 228); Triae_WDR23 LIKE II (SEQ ID NO: 250); Sacof_WDR23 LIKE II (SEQ ID NO: 246); Zeama_WDR23 LIKE II (SEQ ID NO: 256); Orysa_WDR23 LIKE (SEQ ID NO: 238); Triae_WDR23 LIKE (SEQ ID NO: 248); Zeama_WDR23 LIKE (SEQ ID NO: 254); Helan_WDR23 LIKE (SEQ ID NO: 226); Lyces_WDR23 LIKE (SEQ ID NO: 234); Liter_WDR23 LIKE (SEQ ID NO: 232); Glyma_WDR23 LIKE II (SEQ ID NO: 260); Medtr_WDR23 LIKE (SEQ ID NO: 236); Arath_WDR23 LIKE (SEQ ID NO: 216); Linus_WDR23 LIKE (SEQ ID NO: 230); Vitis_WDR23 LIKE (SEQ ID NO: 252); Pinra_WDR23 LIKE (SEQ ID NO: 240); Aqufo_WDR23 LIKE (SEQ ID NO: 218); Poptr_WDR23 LIKE (SEQ ID NO: 242); Homsa_WDR23 (SEQ ID NO: 280); Aspn_LEC14B (SEQ ID NO: 282); Homsa_WDR23 (SEQ ID NO: 281); Aspn_LEC14B (SEQ ID NO: 283); and Consensus (SEQ ID NO: 284).
The present invention will now be described with reference to the following examples, which are by way of illustration alone. The following examples are not intended to completely define or otherwise limit the scope of the invention.
DNA manipulation: unless otherwise stated, recombinant DNA techniques are performed according to standard protocols described in (Sambrook (2001) Molecular Cloning: a laboratory manual, 3rd Edition Cold Spring Harbor Laboratory Press, CSH, New York) or in Volumes 1 and 2 of Ausubel et al. (1994), Current Protocols in Molecular Biology, Current Protocols. Standard materials and methods for plant molecular work are described in Plant Molecular Biology Labfax (1993) by R. D. D. Croy, published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications (UK).
Example 1 Identification of Sequences Related to the Nucleic Acid Sequence Used in the Methods of the InventionSequences (full length cDNA, ESTs or genomic) related to the nucleic acid sequence used in the methods of the present invention were identified amongst those maintained in the Entrez Nucleotides database at the National Center for Biotechnology Information (NCBI) using database sequence search tools, such as the Basic Local Alignment Tool (BLAST) (Altschul et al. (1990) J. Mol. Biol. 215:403-410; and Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402). The program was used to find regions of local similarity between sequences by comparing nucleic acid or polypeptide sequences to sequence databases and by calculating the statistical significance of matches. For example, the polypeptide encoded by the nucleic acid used in the present invention was used for the TBLASTN algorithm, with default settings and the filter to ignore low complexity sequences set off. The output of the analysis was viewed by pairwise comparison, and ranked according to the probability score (E-value), where the score reflect the probability that a particular alignment occurs by chance (the lower the E-value, the more significant the hit). In addition to E-values, comparisons were also scored by percentage identity. Percentage identity refers to the number of identical nucleotides (or amino acids) between the two compared nucleic acid (or polypeptide) sequences over a particular length.
Table A provides a list of nucleic acid sequences related to the nucleic acid sequence used in the methods of the present invention.
The term “table A” used in this specification is to be taken to specify the content of table A1, and/or A2, and/or A3, and/or A4, and/or A5. The term “table A1” used in this specification is to be taken to specify the content of table A1. The term “table A2” used in this specification is to be taken to specify the content of table A2. The term “table A3” used in this specification is to be taken to specify the content of table A3. The term “table A4” used in this specification is to be taken to specify the content of table A4. The term “table A5” used in this specification is to be taken to specify the content of table A5. In one preferred embodiment, the term “table A” means table A1. In one preferred embodiment, the term “table A” means table A2. In one preferred embodiment, the term “table A” means table A3. In one preferred embodiment, the term “table A” means table A4. In one preferred embodiment, the term “table A” means table A5.
The term “table B” used in this specification is to be taken to specify the content of table B1, and/or B2, and/or B3, and/or B4, and/or B5. The term “table B1” used in this specification is to be taken to specify the content of table B1. The term “table B2” used in this specification is to be taken to specify the content of table B2. The term “table B3” used in this specification is to be taken to specify the content of table B3. The term “table B4” used in this specification is to be taken to specify the content of table B4. The term “table B5” used in this specification is to be taken to specify the content of table B5. In one preferred embodiment, the term “table B” means table B1. In one preferred embodiment, the term “table B” means table B2. In one preferred embodiment, the term “table B” means table B3. In one preferred embodiment, the term “table B” means table B4. In one preferred embodiment, the term “table B” means table B5.
The term “table C” used in this specification is to be taken to specify the content of table C1, and/or C2, and/or C3, and/or C4, and/or C5. The term “table C1” used in this specification is to be taken to specify the content of table C1. The term “table C2” used in this specification is to be taken to specify the content of table C2. The term “table C3” used in this specification is to be taken to specify the content of table C3. The term “table C4” used in this specification is to be taken to specify the content of table C4. The term “table C5” used in this specification is to be taken to specify the content of table C5. In one preferred embodiment, the term “table C” means table C1. In one preferred embodiment, the term “table C” means table C2. In one preferred embodiment, the term “table C” means table C3. In one preferred embodiment, the term “table C” means table C4. In one preferred embodiment, the term “table C” means table C5.
The term “table D” used in this specification is to be taken to specify the content of table D1, and/or D2, and/or D3, and/or D4, and/or D5. The term “table D1” used in this specification is to be taken to specify the content of table D1. The term “table D2” used in this specification is to be taken to specify the content of table D2. The term “table D3” used in this specification is to be taken to specify the content of table D3. The term “table D4” used in this specification is to be taken to specify the content of table D4. The term “table D5” used in this specification is to be taken to specify the content of table D5. In one preferred embodiment, the term “table D” means table D1. In one preferred embodiment, the term “table D” means table D2. In one preferred embodiment, the term “table D” means table D3. In one preferred embodiment, the term “table D” means table D4. In one preferred embodiment, the term “table D” means table D5.
Concerning the WDR23-like proteins, in some instances, related sequences have tentatively been assembled and publicly disclosed by research institutions, such as The Institute for Genomic Research (TIGR). The Eukaryotic Gene Orthologs (EGO) database may be used to identify such related sequences, either by keyword search or by using the BLAST algorithm with the nucleic acid sequence or polypeptide sequence of interest. On other instances, special nucleic acid sequence databases have been created for particular organisms, such as by the Joint Genome Institute. In still other instances, the the sequences are obtained after a private, proprietary sequencing and assembly effort has been made, for example for Brassica napus, Helianthus annus and Linum usitatissum.
Example 2 Alignment of PATL PRP38, GATA-Like, ADA2, and WDR23-Like Polypeptide SequencesAlignment of polypeptide sequences was performed using the AlignX programme from the Vector NTI (Invitrogen) which is based on the popular Clustal W algorithm of progressive alignment (Thompson et al. (1997) Nucleic Acids Res 25:4876-4882; Chenna et al. (2003). Nucleic Acids Res 31:3497-3500). Default values were: for the gap open penalty of 10, for the gap extension penalty of 0.1 and the selected weight matrix was Blosum 62 (if polypeptides are aligned). Sequence conservation among PATL polypeptides was higher at the C-terminus than at the N-terminus. A consensus sequence is given. Conserved amino acids are indicated. The PATL polypeptides are aligned in
Sequence conservation among PRP38 polypeptides was essentially in the N-terminal PRP38 domain of the polypeptides, the N-terminal domain usually being more variable in sequence length and composition, was enriched in acidic amino acids. The PRP38 polypeptides are aligned in
Sequence conservation among GATA-like polypeptides is essentially in the GATA domain of the polypeptides and in the C-terminus, the N-terminal domain usually being more variable in sequence length and composition. The GATA-like polypeptides are aligned in
Sequence conservation among ADA2 polypeptides was essentially along the conserved Zn finger ZZ type, SANT DNA binding and SWIRM domains of the polypeptides. A consensus sequence is given. The Zn finger ZZ type domain was enriched in cystein residues. The ADA2 polypeptides are aligned in
Mutliple sequence alignment of the full length WDR23-like polypeptide sequences in Table AS was performed using the AlignX algorithm (from Vector NTI 10.3, Invitrogen Corporation). Results of the alignment are shown in
In
Global percentages of similarity and identity between full length polypeptide sequences useful in performing the methods of the invention were determined using one of the methods available in the art, the MatGAT (Matrix Global Alignment Tool) software (BMC Bioinformatics. 2003 4:29. MatGAT: an application that generates similarity/identity matrices using protein or DNA sequences. Campanella J J, Bitincka L, Smalley J; software hosted by Ledion Bitincka). MatGAT software generates similarity/identity matrices for DNA or protein sequences without needing pre-alignment of the data. The program performs a series of pair-wise alignments using the Myers and Miller global alignment algorithm (with a gap opening penalty of 12, and a gap extension penalty of 2), calculates similarity and identity using for example Blosum 62 (for polypeptides), and then places the results in a distance matrix. Sequence similarity is shown in the bottom half of the dividing line and sequence identity is shown in the top half of the diagonal dividing line.
Parameters used in the comparison were:
Results of the software analysis are shown in Table B1 for the global similarity and identity over the full length of the polypeptide sequences. Percentage identity is given above the diagonal in bold and percentage similarity is given below the diagonal (normal face).
The percentage identity between the PATL polypeptide sequences useful in performing the methods of the invention can be as low as 23.7% amino acid identity compared to Orysa_PATL—1 polypeptide (SEQ ID NO: 2).
Concerning the PRP38 polypeptides, results of the AlignX software analysis are shown in Table B2 for the global identity over the full length of selected polypeptide sequences from Table A2 compared to the Arath_PRP38—1 polypeptide (Table A2).
The percentage identity between the PRP38 polypeptide sequences of Table B2 and useful in performing the methods of the invention can be as low as 23.6% amino acid identity compared to SEQ ID NO: 77.
Concerning the GATA-like proteins, parameters used in the comparison were:
Results of the software analysis are shown in Table B3 for the global similarity and identity over the full length of the polypeptide sequences. Percentage identity is given above the diagonal in bold and percentage similarity is given below the diagonal (normal face).
The percentage identity between the GATA-like polypeptide sequences useful in performing the methods of the invention can be as low as 14% amino acid identity compared to SEQ ID NO: 129.
Concerning the ADA2 polypeptides, parameters used in the comparison were:
Results of the software analysis are shown in Table B4 for the global similarity and identity over the full length of the polypeptide sequences. Percentage identity is given above the diagonal in bold and percentage similarity is given below the diagonal (normal face).
The percentage identity between the ADA2 polypeptide sequences useful in performing the methods of the invention can be as low as 21.2% amino acid identity compared to Arath_ADA2—1 polypeptide (SEQ ID NO: 182).
Concerning the WDR23-like proteins, parameters used in the comparison were:
-
- Scoring matrix: Blosum62
- First Gap: 12
- Extending gap: 2
Results of the software analysis are shown in Table B5 for the global similarity and identity over the full length of the polypeptide sequences (excluding the partial polypeptide sequences).
The same analysis was done between the Conserved Domain (CD) as represented by SEQ ID NO: 271 (and comprised in SEQ ID NO: 216), and the Conserved Domain of the full length polypeptides of Table A5 (as highlighted in
The percentage identity between the full length polypeptide sequences useful in performing the methods of the invention can be as low as 54% amino acid identity compared to SEQ ID NO: 216.
The percentage identity between the Conserved Domain (CD) as represented by SEQ ID NO: 271 (and comprised in SEQ ID NO: 216), and the Conserved Domain of the polypeptides of Table A5 (as highlighted in
Conserved domain proteins were identified by searching the InterPro database. The Integrated Resource of Protein Families, Domains and Sites (InterPro) database is an integrated interface for the commonly used signature databases for text- and sequence-based searches. The InterPro database combines these databases, which use different methodologies and varying degrees of biological information about well-characterized proteins to derive protein signatures. Collaborating databases include SWISS-PROT, PROSITE, TrEMBL, Panther, PRINTS, ProDom and Pfam, Smart and TIGRFAMs. Pfam is a large collection of multiple sequence alignments and hidden Markov models covering many common protein domains and families. Pfam is hosted at the Sanger Institute server in the United Kingdom. Interpro is hosted at the European Bioinformatics Institute in the United Kingdom.
The results of the InterPro scan of the polypeptide sequence as represented by SEQ ID NO: 2 are presented in Table C5.
The results of the Pfam scan of the polypeptide sequence as represented by SEQ ID NO: 77 are presented in Table C2.
The results of the InterPro scan of the polypeptide sequence as represented by SEQ ID NO: 129 are presented in Table C3.
The results of the InterPro scan of the polypeptide sequence as represented by SEQ ID NO: 182 are presented in Table C4.
The results of the InterPro scan of the polypeptide sequence as represented by SEQ ID NO: 216 are presented in Table C5.
The nucleic acid sequence used in the methods of the invention was amplified by PCR using as template a custom-made Arabidopsis thaliana seedlings cDNA library (in pCMV Sport 6.0; Invitrogen, Paisley, UK). PCR was performed using Hifi Taq DNA polymerase in standard conditions, using 200 ng of template in a 50 μl PCR mix.
Concerning PATL, the primers used were SEQ ID NO: 73: 5′-ggggacaagtttgtacaaaaaagcaggcttaaacaatggcggaggagccac-3′ and SEQ ID NO: 74; 5′-ggggaccactttgtacaagaaagctgggtgtggtgaatctggtgatcagg-3′, which include the AttB sites for Gateway recombination. The amplified PCR fragment was purified also using standard methods. The first step of the Gateway procedure, the BP reaction, was then performed, during which the PCR fragment recombines in vivo with the pDONR201 plasmid to produce, according to the Gateway terminology, an “entry clone”, pOrysa_PATL—1. Plasmid pDONR201 was purchased from Invitrogen, as part of the Gateway® technology.
The entry clone comprising SEQ ID NO: 1 was then used in an LR reaction with two destination vector used for Oryza sativa transformation. A first vector contained as functional elements within the T-DNA borders: a plant selectable marker; a screenable marker expression cassette; and a Gateway cassette intended for LR in vivo recombination with the nucleic acid sequence of interest already cloned in the entry clone. A rice GOS2 promoter (SEQ ID NO: 75) for constitutive expression was located upstream of this Gateway cassette.
After the LR recombination step, the resulting expression vectors pGOS2::Orysa_PATL—1 (
Concerning the PRP38, the primers used were SEQ ID NO: 125: 5′ ggggacaagtttgtacaaaaaagcaggcttaaacaatggcggagatacagtcaaa 3′ and SEQ ID NO: 126; 5′ ggggaccactttgtacaagaaagctgggttcacctccaagaggaacca 3′, which include the AttB sites for Gateway recombination. The amplified PCR fragment was purified also using standard methods. The first step of the Gateway procedure, the BP reaction, was then performed, during which the PCR fragment recombines in vivo with the pDONR201 plasmid to produce, according to the Gateway terminology, an “entry clone”, pPRP38. Plasmid pDONR201 was purchased from Invitrogen, as part of the Gateway® technology.
The entry clone comprising SEQ ID NO: 76 was then used in an LR reaction with a destination vector used for Oryza sativa transformation. This vector contained as functional elements within the T-DNA borders: a plant selectable marker; a screenable marker expression cassette; and a Gateway cassette intended for LR in vivo recombination with the nucleic acid sequence of interest already cloned in the entry clone. A rice GOS2 promoter (SEQ ID NO: 127) for constitutive expression was located upstream of this Gateway cassette.
After the LR recombination step, the resulting expression vector pGOS2::PRP38 (
which include the AttB sites for Gateway recombination. The amplified PCR fragment was purified also using standard methods. The first step of the Gateway procedure, the BP reaction, was then performed, during which the PCR fragment recombines in vivo with the pDONR201 plasmid to produce, according to the Gateway terminology, an “entry clone”, pGATA-like. Plasmid pDONR201 was purchased from Invitrogen, as part of the Gateway® technology.
The entry clone comprising SEQ ID NO: 128 was then used in an LR reaction with a destination vector used for Oryza sativa transformation. This vector contained as functional elements within the T-DNA borders: a plant selectable marker; a screenable marker expression cassette; and a Gateway cassette intended for LR in vivo recombination with the nucleic acid sequence of interest already cloned in the entry clone. A rice GOS2 promoter (SEQ ID NO: 135) for root specific expression was located upstream of this Gateway cassette.
After the LR recombination step, the resulting expression vector pGOS2::GATA-like (
The nucleic acid sequence of SEQ ID NO: 177, used in the methods of the invention was amplified by PCR using as template a custom-made Oryza sativa seedlings cDNA library (in pCMV Sport 6.0; Invitrogen, Paisley, UK). PCR was performed using Hifi Taq DNA polymerase in standard conditions, using 200 ng of template in a 50 μl PCR mix. The primers used were prm10106 (SEQ ID NO: 179; sense, start codon in bold): 5′-ggggacaagt ttgtacaaaaaagcaggcttaaacaatgcttcaccattactacagc-3′ and prm10107 (SEQ ID NO: 180; reverse, complementary): 5′-ggggaccactttgtacaagaaagctgggtcca acgctaatgctacact-3′, which include the AttB sites for Gateway recombination. The amplified PCR fragment was purified also using standard methods. The further cloning procedure was as described above.
Concerning ADA2, the primers used were SEQ ID NO: 211: 5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTAAACAATGGGTCGTTCGAAACTAGC-3′ and SEQ ID NO: 212; 5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCATGTTAGGACCATGAAGCT ATG-3′, which include the AttB sites for Gateway recombination. The amplified PCR fragment was purified also using standard methods. The first step of the Gateway procedure, the BP reaction, was then performed, during which the PCR fragment recombines in vivo with the pDONR201 plasmid to produce, according to the Gateway terminology, an “entry clone”, pAtADA2—1. Plasmid pDONR201 was purchased from Invitrogen, as part of the Gateway® technology.
The entry clone comprising SEQ ID NO: 181 was then used in an LR reaction with two destination vector used for Oryza sativa transformation. A first vector contained as functional elements within the T-DNA borders: a plant selectable marker; a screenable marker expression cassette; and a Gateway cassette intended for LR in vivo recombination with the nucleic acid sequence of interest already cloned in the entry clone. A rice HMGP promoter (SEQ ID NO: 213) for constitutive expression was located upstream of this Gateway cassette. On a second vector having same functional elements within the T-DNA borders as described above, a rice EXP9 promoter (SEQ ID NO: 214) was located upstream of the Gateway cassette.
After the LR recombination step, the resulting expression vectors pHMG::Arath_ADA2—1 (
Unless otherwise stated, recombinant DNA techniques are performed according to standard protocols described in (Sambrook (2001) Molecular Cloning: a laboratory manual, 3rd Edition Cold Spring Harbor Laboratory Press, CSH, New York) or in Volumes 1 and 2 of Ausubel et al. (1994), Current Protocols in Molecular Biology, Current Protocols. Standard materials and methods for plant molecular work are described in Plant Molecular Biology Labfax (1993) by R. D. D. Croy, published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications (UK).
Concerning the WDR23-like polypeptide, the Arabidopsis thaliana cDNA encoding a WDR23-like polypeptide sequence as represented by SEQ ID NO: 216 was amplified by PCR using as template cDNA synthesized from mRNA extracted from different tissues of Arabidopsis thaliana grown under different conditions. The following primers, which include the AttB sites for Gateway recombination, were used for PCR amplification:
PCR was performed using Hifi Taq DNA polymerase in standard conditions. A PCR fragment of the expected length (including attB sites) was amplified and purified also using standard methods. The first step of the Gateway procedure, the BP reaction, was then performed, during which the PCR fragment recombined in vivo with the pDONR201 plasmid to produce, according to the Gateway terminology, an “entry clone”. Plasmid pDONR201 was purchased from Invitrogen, as part of the Gateway® technology.
Example 5A Topology Prediction of the Polypeptide Sequences Useful in Performing the Methods of the InventionTargetP 1.1 predicts the subcellular location of eukaryotic proteins. The location assignment is based on the predicted presence of any of the N-terminal pre-sequences: chloroplast transit peptide (cTP), mitochondrial targeting peptide (mTP) or secretory pathway signal peptide (SP). Scores on which the final prediction is based are not really probabilities, and they do not necessarily add to one. However, the location with the highest score is the most likely according to TargetP, and the relationship between the scores (the reliability class) may be an indication of how certain the prediction is. The reliability class (RC) ranges from 1 to 5, where 1 indicates the strongest prediction. TargetP is maintained at the server of the Technical University of Denmark.
For the sequences predicted to contain an N-terminal presequence a potential cleavage site can also be predicted.
A number of parameters were selected, such as organism group (non-plant or plant), cutoff sets (none, predefined set of cutoffs, or user-specified set of cutoffs), and the calculation of prediction of cleavage sites (yes or no).
The results of TargetP 1.1 analysis of the polypeptide sequence as represented by SEQ ID NO: 129 are presented Table D3. The “plant” organism group has been selected, no cutoffs defined, and the predicted length of the transit peptide requested. The subcellular localization of the polypeptide sequence as represented by SEQ ID NO: 129 may be the cytoplasm or nucleus, no transit peptide is predicted.
Many other algorithms can be used to perform such analyses, including:
-
- ChloroP 1.1 hosted on the server of the Technical University of Denmark;
- Protein Prowler Subcellular Localisation Predictor version 1.2 hosted on the server of the Institute for Molecular Bioscience, University of Queensland, Brisbane, Australia;
- PENCE Proteome Analyst PA-GOSUB 2.5 hosted on the server of the University of Alberta, Edmonton, Alberta, Canada;
- TMHMM, hosted on the server of the Technical University of Denmark
The Agrobacterium containing the expression vector was used to transform Oryza sativa plants. Mature dry seeds of the rice japonica cultivar Nipponbare were dehusked. Sterilization was carried out by incubating for one minute in 70% ethanol, followed by 30 minutes in 0.2% HgCl2, followed by a 6 times 15 minutes wash with sterile distilled water. The sterile seeds were then germinated on a medium containing 2,4-D (callus induction medium). After incubation in the dark for four weeks, embryogenic, scutellum-derived calli were excised and propagated on the same medium. After two weeks, the calli were multiplied or propagated by subculture on the same medium for another 2 weeks. Embryogenic callus pieces were sub-cultured on fresh medium 3 days before co-cultivation (to boost cell division activity).
Agrobacterium strain LBA4404 containing the expression vector was used for co-cultivation. Agrobacterium was inoculated on AB medium with the appropriate antibiotics and cultured for 3 days at 28° C. The bacteria were then collected and suspended in liquid co-cultivation medium to a density (OD600) of about 1. The suspension was then transferred to a Petri dish and the calli immersed in the suspension for 15 minutes. The callus tissues were then blotted dry on a filter paper and transferred to solidified, co-cultivation medium and incubated for 3 days in the dark at 25° C. Co-cultivated calli were grown on 2,4-D-containing medium for 4 weeks in the dark at 28° C. in the presence of a selection agent. During this period, rapidly growing resistant callus islands developed. After transfer of this material to a regeneration medium and incubation in the light, the embryogenic potential was released and shoots developed in the next four to five weeks. Shoots were excised from the calli and incubated for 2 to 3 weeks on an auxin-containing medium from which they were transferred to soil. Hardened shoots were grown under high humidity and short days in a greenhouse.
Approximately 35 independent T0 rice transformants were generated for one construct. The primary transformants were transferred from a tissue culture chamber to a greenhouse. After a quantitative PCR analysis to verify copy number of the T-DNA insert, only single copy transgenic plants that exhibit tolerance to the selection agent were kept for harvest of T1 seed. Seeds were then harvested three to five months after transplanting. The method yielded single locus transformants at a rate of over 50% (Aldemita and Hodges 1996, Chan et al. 1993, Hiei et al. 1994).
Corn TransformationTransformation of maize (Zea mays) is performed with a modification of the method described by Ishida et al. (1996) Nature Biotech 14(6): 745-50. Transformation is genotype-dependent in corn and only specific genotypes are amenable to transformation and regeneration. The inbred line A188 (University of Minnesota) or hybrids with A188 as a parent are good sources of donor material for transformation, but other genotypes can be used successfully as well. Ears are harvested from corn plant approximately 11 days after pollination (DAP) when the length of the immature embryo is about 1 to 1.2 mm. Immature embryos are cocultivated with Agrobacterium tumefaciens containing the expression vector, and transgenic plants are recovered through organogenesis. Excised embryos are grown on callus induction medium, then maize regeneration medium, containing the selection agent (for example imidazolinone but various selection markers can be used). The Petri plates are incubated in the light at 25° C. for 2-3 weeks, or until shoots develop. The green shoots are transferred from each embryo to maize rooting medium and incubated at 25° C. for 2-3 weeks, until roots develop. The rooted shoots are transplanted to soil in the greenhouse. T1 seeds are produced from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.
Wheat TransformationTransformation of wheat is performed with the method described by Ishida et al. (1996) Nature Biotech 14(6): 745-50. The cultivar Bobwhite (available from CIMMYT, Mexico) is commonly used in transformation. Immature embryos are co-cultivated with Agrobacterium tumefaciens containing the expression vector, and transgenic plants are recovered through organogenesis. After incubation with Agrobacterium, the embryos are grown in vitro on callus induction medium, then regeneration medium, containing the selection agent (for example imidazolinone but various selection markers can be used). The Petri plates are incubated in the light at 25° C. for 2-3 weeks, or until shoots develop. The green shoots are transferred from each embryo to rooting medium and incubated at 25° C. for 2-3 weeks, until roots develop. The rooted shoots are transplanted to soil in the greenhouse. T1 seeds are produced from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.
Soybean Transformation
Soybean is transformed according to a modification of the method described in the Texas A&M patent U.S. Pat. No. 5,164,310. Several commercial soybean varieties are amenable to transformation by this method. The cultivar Jack (available from the Illinois Seed foundation) is commonly used for transformation. Soybean seeds are sterilised for in vitro sowing. The hypocotyl, the radicle and one cotyledon are excised from seven-day old young seedlings. The epicotyl and the remaining cotyledon are further grown to develop axillary nodes. These axillary nodes are excised and incubated with Agrobacterium tumefaciens containing the expression vector. After the cocultivation treatment, the explants are washed and transferred to selection media. Regenerated shoots are excised and placed on a shoot elongation medium. Shoots no longer than 1 cm are placed on rooting medium until roots develop. The rooted shoots are transplanted to soil in the greenhouse. T1 seeds are produced from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.
Rapeseed/Canola TransformationCotyledonary petioles and hypocotyls of 5-6 day old young seedling are used as explants for tissue culture and transformed according to Babic et al. (1998, Plant Cell Rep 17: 183-188). The commercial cultivar Westar (Agriculture Canada) is the standard variety used for transformation, but other varieties can also be used. Canola seeds are surface-sterilized for in vitro sowing. The cotyledon petiole explants with the cotyledon attached are excised from the in vitro seedlings, and inoculated with Agrobacterium (containing the expression vector) by dipping the cut end of the petiole explant into the bacterial suspension. The explants are then cultured for 2 days on MSBAP-3 medium containing 3 mg/l BAP, 3% sucrose, 0.7% Phytagar at 23° C., 16 hr light. After two days of co-cultivation with Agrobacterium, the petiole explants are transferred to MSBAP-3 medium containing 3 mg/l BAP, cefotaxime, carbenicillin, or timentin (300 mg/l) for 7 days, and then cultured on MSBAP-3 medium with cefotaxime, carbenicillin, or timentin and selection agent until shoot regeneration. When the shoots are 5-10 mm in length, they are cut and transferred to shoot elongation medium (MSBAP-0.5, containing 0.5 mg/l BAP). Shoots of about 2 cm in length are transferred to the rooting medium (MS0) for root induction. The rooted shoots are transplanted to soil in the greenhouse. T1 seeds are produced from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.
Alfalfa TransformationA regenerating clone of alfalfa (Medicago sativa) is transformed using the method of (McKersie et al., 1999 Plant Physiol 119: 839-847). Regeneration and transformation of alfalfa is genotype dependent and therefore a regenerating plant is required. Methods to obtain regenerating plants have been described. For example, these can be selected from the cultivar Rangelander (Agriculture Canada) or any other commercial alfalfa variety as described by Brown D C W and A Atanassov (1985. Plant Cell Tissue Organ Culture 4: 111-112). Alternatively, the RA3 variety (University of Wisconsin) has been selected for use in tissue culture (Walker et al., 1978 Am J Bot 65:654-659). Petiole explants are cocultivated with an overnight culture of Agrobacterium tumefaciens C58C1 pMP90 (McKersie et al., 1999 Plant Physiol 119: 839-847) or LBA4404 containing the expression vector. The explants are cocultivated for 3 d in the dark on SH induction medium containing 288 mg/L Pro, 53 mg/L thioproline, 4.35 g/L K2SO4, and 100 μm acetosyringinone. The explants are washed in half-strength Murashige-Skoog medium (Murashige and Skoog, 1962) and plated on the same SH induction medium without acetosyringinone but with a suitable selection agent and suitable antibiotic to inhibit Agrobacterium growth. After several weeks, somatic embryos are transferred to BOi2Y development medium containing no growth regulators, no antibiotics, and 50 g/L sucrose. Somatic embryos are subsequently germinated on half-strength Murashige-Skoog medium. Rooted seedlings were transplanted into pots and grown in a greenhouse. T1 seeds are produced from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.
Cotton TransformationCotton is transformed using Agrobacterium tumefaciens according to the method described in U.S. Pat. No. 5,159,135. Cotton seeds are surface sterilised in 3% sodium hypochlorite solution during 20 minutes and washed in distilled water with 500 μg/ml cefotaxime. The seeds are then transferred to SH-medium with 50 μg/ml benomyl for germination. Hypocotyls of 4 to 6 days old seedlings are removed, cut into 0.5 cm pieces and are placed on 0.8% agar. An Agrobacterium suspension (approx. 108 cells per ml, diluted from an overnight culture transformed with the gene of interest and suitable selection markers) is used for inoculation of the hypocotyl explants. After 3 days at room temperature and lighting, the tissues are transferred to a solid medium (1.6 g/I Gelrite) with Murashige and Skoog salts with B5 vitamins (Gamborg et al., Exp. Cell Res. 50:151-158 (1968)), 0.1 mg/l 2,4-D, 0.1 mg/l 6-furfurylaminopurine and 750 μg/ml MgCL2, and with 50 to 100 μg/ml cefotaxime and 400-500 μg/ml carbenicillin to kill residual bacteria. Individual cell lines are isolated after two to three months (with subcultures every four to six weeks) and are further cultivated on selective medium for tissue amplification (30° C., 16 hr photoperiod). Transformed tissues are subsequently further cultivated on non-selective medium during 2 to 3 months to give rise to somatic embryos. Healthy looking embryos of at least 4 mm length are transferred to tubes with SH medium in fine vermiculite, supplemented with 0.1 mg/l indole acetic acid, 6 furfurylaminopurine and gibberellic acid. The embryos are cultivated at 30° C. with a photoperiod of 16 hrs, and plantlets at the 2 to 3 leaf stage are transferred to pots with vermiculite and nutrients. The plants are hardened and subsequently moved to the greenhouse for further cultivation.
Concerning WDR23-like genes, Cotton (Gossypium hirsutum L.) transformation is performed using Agrobacterium tumefaciens, on hypocotyls explants. The commercial cultivars such as Coker 130 or Coker 312 (SeedCo, Lubbock, Tex.) are standard varieties used for transformation, but other varieties can also be used. The seeds are surface sterilized and germinated in the dark. Hypocotyl explants are cut from the germinated seedlings to lengths of about 1-1.5 centimeter. The hypotocyl explant is submersed in the Agrobacterium tumefaciens inoculum containing the expression vector, for 5 minutes then co-cultivated for about 48 hours on MS+1.8 mg/l KNO3+2% glucose at 24° C., in the dark. The explants are transferred the same medium containing appropriate bacterial and plant selectable markers (renewed several times), until embryogenic calli is seen. The calli are separated and subcultured until somatic embryos appear. Plantlets derived from the somatic embryos are matured on rooting medium until roots develop. The rooted shoots are transplanted to potting soil in the greenhouse. T1 seeds are produced from plants that exhibit tolerance to the selection agent and that contain a single copy of the T-DNA insert.
Example 6B Expression Vector Construction Using the Nucleic Acid Sequence as Represented by SEQ ID NO: 215The entry clone comprising SEQ ID NO: 215 was subsequently used in an LR reaction with a destination vector used for Oryza sativa transformation. This vector contained as functional elements within the T-DNA borders: a plant selectable marker; a screenable marker expression cassette; and a Gateway cassette intended for LR in vivo recombination with the nucleic acid sequence of interest already cloned in the entry clone. A rice GOS2 promoter (SEQ ID NO: 272) for constitutive expression was located upstream of this Gateway cassette. A second destination vector for Oryza sativa transformation was also produced, with a rice metallothionein promoter (MT; SEQ ID NO: 273) for meristem-specific expression.
After the LR recombination step, the resulting expression vector pGOS2::WDR23-like and pMT::WDR23-like (
7.1 Evaluation Setup
Approximately 35 independent T0 rice transformants were generated. The primary transformants were transferred from a tissue culture chamber to a greenhouse for growing and harvest of T1 seed. Six events, of which the T1 progeny segregated 3:1 for presence/absence of the transgene, were retained. For each of these events, approximately 10 T1 seedlings containing the transgene (hetero- and homo-zygotes) and approximately 10 T1 seedlings lacking the transgene (nullizygotes) were selected by monitoring visual marker expression. The transgenic plants and the corresponding nullizygotes were grown side-by-side at random positions. Greenhouse conditions (non-stress conditions) were of shorts days (12 hours light), 28° C. in the light and 22° C. in the dark, and a relative humidity of 70%. Frequent watering was applied to satisfy plant water and nutrient needs in order to grow and develop with a healthy appearance.
From the stage of sowing until the stage of maturity the plants were passed several times through a digital imaging cabinet. At each time point digital images (2048×1536 pixels, 16 million colours) were taken of each plant from at least 6 different angles.
Drought ScreenPlants from T2 seeds are grown in potting soil under normal conditions until they approached the heading stage. They were then transferred to a “dry” section where irrigation was withheld. Humidity probes were inserted in randomly chosen pots to monitor the soil water content (SWC). When SWC went below certain thresholds, the plants were automatically re-watered continuously until a normal level was reached again. The plants were then re-transferred again to normal conditions. The rest of the cultivation (plant maturation, seed harvest) was the same as for plants not grown under abiotic stress conditions. Growth and yield parameters are recorded as detailed for growth under normal conditions.
Nitrogen Use Efficiency ScreenRice plants from T2 seeds are grown in potting soil under normal conditions except for the nutrient solution. The pots were watered from transplantation to maturation with a specific nutrient solution containing reduced N nitrogen (N) content, usually between 7 to 8 times less. The rest of the cultivation (plant maturation, seed harvest) was the same as for plants not grown under abiotic stress. Growth and yield parameters are recorded as detailed for growth under normal conditions.
7.2 Statistical Analysis: F Test
A two factor ANOVA (analysis of variants) was used as a statistical model for the overall evaluation of plant phenotypic characteristics. An F test was carried out on all the parameters measured of all the plants of all the events transformed with the gene of the present invention. The F test was carried out to check for an effect of the gene over all the transformation events and to verify for an overall effect of the gene, also known as a global gene effect. The threshold for significance for a true global gene effect was set at a 5% probability level for the F test. A significant F test value points to a gene effect, meaning that it is not only the mere presence or position of the gene that is causing the differences in phenotype.
Because two experiments with overlapping events were carried out, a combined analysis was performed. This is useful to check consistency of the effects over the two experiments, and if this is the case, to accumulate evidence from both experiments in order to increase confidence in the conclusion. The method used was a mixed-model approach that takes into account the multilevel structure of the data (i.e. experiment-event-segregants). P values were obtained by comparing likelihood ratio test to chi square distributions.
7.3 Parameters Measured
Biomass-Related Parameter MeasurementFrom the stage of sowing until the stage of maturity the plants were passed several times through a digital imaging cabinet. At each time point digital images (2048×1536 pixels, 16 million colours) were taken of each plant from at least 6 different angles.
The plant aboveground area (or leafy biomass) was determined by counting the total number of pixels on the digital images from aboveground plant parts discriminated from the background. This value was averaged for the pictures taken on the same time point from the different angles and was converted to a physical surface value expressed in square mm by calibration. Experiments show that the aboveground plant area measured this way correlates with the biomass of plant parts above ground. The above ground area is the area measured at the time point at which the plant had reached its maximal leafy biomass. The early vigour is the plant (seedling) aboveground area three weeks post-germination. Increase in root biomass is expressed as an increase in total root biomass (measured as maximum biomass of roots observed during the lifespan of a plant); or as an increase in the root/shoot index (measured as the ratio between root mass and shoot mass in the period of active growth of root and shoot).
Early vigour was determined by counting the total number of pixels from aboveground plant parts discriminated from the background. This value was averaged for the pictures taken on the same time point from different angles and was converted to a physical surface value expressed in square mm by calibration. The results described below are for plants three weeks post-germination.
Seed-Related Parameter MeasurementsThe mature primary panicles were harvested, counted, bagged, barcode-labelled and then dried for three days in an oven at 37° C. The panicles were then threshed and all the seeds were collected and counted. The filled husks were separated from the empty ones using an air-blowing device. The empty husks were discarded and the remaining fraction was counted again. The filled husks were weighed on an analytical balance. The number of filled seeds was determined by counting the number of filled husks that remained after the separation step. The total seed yield was measured by weighing all filled husks harvested from a plant. Total seed number per plant was measured by counting the number of husks harvested from a plant. Thousand Kernel Weight (TKW) is extrapolated from the number of filled seeds counted and their total weight. The Harvest Index (HI) in the present invention is defined as the ratio between the total seed yield and the above ground area (mm2), multiplied by a factor 106. The total number of flowers per panicle as defined in the present invention is the ratio between the total number of seeds and the number of mature primary panicles. The seed fill rate as defined in the present invention is the proportion (expressed as a %) of the number of filled seeds over the total number of seeds (or florets).
Salt Stress ScreenPlants are grown on a substrate made of coco fibers and argex (3 to 1 ratio). A normal nutrient solution is used during the first two weeks after transplanting the plantlets in the greenhouse. After the first two weeks, 25 mM of salt (NaCl) is added to the nutrient solution, until the plants were harvested. Growth and yield parameters are recorded as detailed for growth under normal conditions.
Reduced Nutrient (Nitrogen) Availability ScreenPlants from six events (T2 seeds) are grown in potting soil under normal conditions except for the nutrient solution. The pots are watered from transplantation to maturation with a specific nutrient solution containing reduced N nitrogen (N) content, usually between 7 to 8 times less. The rest of the cultivation (plant maturation, seed harvest) is the same as for plants not grown under abiotic stress. Growth and yield parameters are recorded as detailed for growth under normal conditions.
Example 8 Results of the Phenotypic Evaluation of the Transgenic PlantsThe results of the evaluation of transgenic rice plants expressing Orysa_PATL—1 (SEQ ID NO:1) nucleic acid from the GOS2 promoter and grown in a green house under non-stress conditions (Example 7) are presented below. An increase of at least 3% was observed for the seed weight (total seed weight), number of filled seeds, and plant height (Table D1).
The results of the evaluation of transgenic rice plants expressing a AtPRP38—1 (SEQ ID NO:76) nucleic acid grown in a green house under non-stress conditions (Example 7) are presented below. An increase of at least 5% was observed for aboveground biomass (AreaMax), emergence vigour (early vigour), total seed yield, number of filled seeds, fill rate, harvest index, and number of seeds per plant (Table D2).
Evaluation of transgenic rice plants expressing the GA TA-like nucleic acid represented by SEQ ID NO: 128 under non-stress conditions revealed an increase of Thousand Kernel Weight (all six events, overall increase of 9.1%, p-value: 0.00001). No significant changes were observed for other yield parameters.
Evaluation of transgenic rice plants expressing the GATA-like nucleic acid represented by SEQ ID NO: 177 under non-stress conditions revealed an increase of total weight of seeds (four lines out of six, overall increase of 16.9%, p-value: 0.00001) and number of filled seeds (four lines out of six, overall increase of 16.0%, p-value: 0.00001). No significant changes were observed for other yield parameters.
The results of the evaluation of transgenic rice plants expressing Arath_ADA2—1 (SEQ ID NO: 181) nucleic acid from either the pHMG or the pEXP promoters and grown in a green house under non-stress conditions (Example 7) are presented below. An increase of at least 3% was observed for the seed weight (total seed weight), number of filled seeds, seed filling (fill rate), number of flowers per planicle and harvest index (Table D4).
The results of the evaluation of T1 and T2 generation transgenic rice plants expressing the nucleic acid sequence encoding a WDR23-like polypeptide as represented by SEQ ID NO: 216, under the control of a GOS2 promoter for constitutive expression, are presented below.
There was a significant increase in the total seed yield per plant, in the seed filling rate, in the number of filled seeds, in the harvest index, and in the Thousand Kernel Weight (TKW) of the transgenic plants compared to corresponding nullizygotes (controls), as shown in Table D5, in both T1 and T2 generation phenotypic analysis.
The results of the evaluation of T1 and T2 generation transgenic rice plants expressing the nucleic acid sequence encoding a WDR23-like polypeptide as represented by SEQ ID NO: 216, under the control of a metallothionein MT promoter for meristem-specific expression, are presented below.
There was a significant increase in the total seed yield per plant, in the seed filling rate, in the number of filled seeds, in the harvest index, and in the Thousand Kernel Weight (TKW) of the transgenic plants compared to corresponding nullizygotes (controls), as shown in Table E, in both T1 and T2 generation phenotypic analysis.
Claims
1. A method for enhancing yield-related traits in plants relative to control plants, comprising modulating expression in a plant of a nucleic acid encoding:
- a) a PRP38 polypeptide,
- b) an ADA2 polypeptide and optionally selecting for plants having enhanced yield-related traits,
- c) a GATA-like polypeptide, wherein said GATA-like polypeptide belongs to subfamily II of GATA transcription factors and comprises a GATA domain, or
- d) a WDR23-like polypeptide, which WDR23-like polypeptide comprises a domain having at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more sequence identity to a Conserved Domain (CD) comprising the amino acid sequence of SEQ ID NO: 271, and optionally selecting for plants having increased yield-related traits.
2. The method of claim 1, wherein said GATA-like polypeptide comprises one or more of the following motifs:
- (i) a Motif 1c comprising the amino acid sequence of SEQ ID NO: 130;
- (ii) a Motif 2c comprising the amino acid sequence of SEQ ID NO: 131;
- (iii) a Motif 3c comprising the amino acid sequence of SEQ ID NO: 132.
3. The method of claim 1, wherein said modulated expression is effected by introducing and expressing in a plant a nucleic acid encoding a PRP38 polypeptide, a nucleic acid encoding a GATA-like polypeptide, a nucleic acid encoding an ADA2 polypeptide, or a nucleic acid encoding a WDR23-like polypeptide.
4. The method of claim 1, wherein said nucleic acid encoding a PRP38, ADA2, GATA-like or a WDR23-like polypeptide encodes any one of the proteins listed in Table A or is a portion of such a nucleic acid, or a nucleic acid capable of hybridizing with such a nucleic acid.
5. The method of claim 1, wherein said nucleic acid sequence encodes an orthologue or paralogue of any of the proteins given in Table A.
6. The method of claim 1, wherein said enhanced yield-related traits comprise increased yield, thousand kernel weight, or seed yield relative to control plants.
7. The method of claim 1, wherein said enhanced yield-related traits are obtained under conditions of nitrogen deficiency.
8. The method of claim 1, wherein said enhanced yield-related traits are obtained non-stress conditions.
9. The method of claim 1, wherein said nucleic acid is operably linked to a constitutive promoter, a GOS2 promoter, or a GOS2 promoter from rice.
10. The method of claim 1, wherein said nucleic acid encoding GATA-like polypeptide is of plant origin, from a monocotyledonous plant, from a plant of the family Poaceae, from a plant of the genus Oryza, or from an Oryza sativa plant.
11. A plant obtained by the method of claim 1, or a part, seed or progeny thereof, wherein said plant, or said part, seed or progeny thereof, comprises a recombinant nucleic acid encoding a PRP38, ADA2, GATA-like or a WDR23-like polypeptide.
12. A construct comprising:
- (i) a nucleic acid encoding a PRP38, ADA2, GATA-like or a WDR23-like polypeptide as defined in claim 1;
- (ii) one or more control sequences capable of driving expression of the nucleic acid of (i); and optionally
- (iii) a transcription termination sequence.
13. A method for making plants having increased yield, increased biomass and/or increased seed yield relative to control plants comprising transforming a plant cell, plant or part thereof with the construct of claim 12.
14. A plant, plant part or plant cell transformed with the construct of claim 12.
15. A method for the production of a transgenic plant having increased yield or increased seed yield relative to control plants, comprising:
- (i) introducing and expressing in a plant a nucleic acid encoding a PRP38, ADA2, GATA-like or a WDR23-like polypeptide as defined in claim 1; and
- (ii) cultivating the plant cell under conditions promoting plant growth and development; and optionally
- (iii) selecting for plants having enhanced yield-related traits
16. A transgenic plant having increased yield or increased seed yield relative to control plants, resulting from modulated expression of a nucleic acid encoding a PRP38, ADA2, GATA-like or a WDR23-like polypeptide as defined in claim 1, or a transgenic plant cell derived from said transgenic plant.
17. The transgenic plant of claim 16, or a transgenic plant cell derived thereof, wherein said plant is a crop plant, a monocot, or a cereal, or wherein said plant is rice, maize, wheat, barley, millet, rye, triticale, sorghum, or oats.
18. Harvestable parts of the plant of claim 16, wherein said harvestable parts are shoot biomass and/or seeds.
19. Products derived from the plant of claim 16 and/or from harvestable parts of said plant.
Type: Application
Filed: Feb 7, 2014
Publication Date: Aug 21, 2014
Applicant: BASF Plant Science GmbH (Ludwigshafen)
Inventors: Ana Isabel Sanz Molinero (Madrid), Christophe Reuzeau (La Chapelle Gonaguet), Valerie Frankard (Waterloo), Ramon Serrano Salom (Valencia), José Miguel Mulet Salort (Valencia)
Application Number: 14/175,402
International Classification: C12N 15/82 (20060101);
