PLANTS WITH INCREASED SEED SIZE
The invention relates to genetically modified plants with an altered seed phenotype, in particular increased seed size. The invention relates to a plant that does not produce a functional NGAL2 polypeptide or functional NGAL2 and NGAL3 polypeptides. NGAL2 and NGAL3 are members of the RAV family and comprise a B3 DNA-binding domain and a transcriptional repression motif.
This is a continuation application of U.S. Ser. No. 15/548,398 filed Aug. 2, 2017, which is a National Phase application claiming priority to PCT/GB2016/050245, filed Feb. 3, 2016, which claims priority to PCT/CN2015/072143, filed Feb. 3, 2015, all of which are herein incorporated by reference in their entirety.
FIELD OF THE INVENTIONThe invention relates to transgenic plants with improved growth and yield-related traits, in particular increased seed size. Also within the scope of the invention are related methods, uses, isolated nucleic acids and vector constructs.
INTRODUCTIONThe ever-increasing world population and the dwindling supply of arable land available for agriculture fuels research towards increasing the efficiency of agriculture and providing food security. 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, including increased yield. There are a number of methods that can be used, for example genome editing (using CRISPR or TALEN) or mutagenesis.
A trait of particular economic interest is increased seed size. Seed size is an important agronomic trait which increased crop yield, and is also a key ecological trait that influences many aspects of a species' regeneration strategy, such as seedling survival rates and seed dispersal syndrome (Harper et al., 1970; Westoby et al., 2002; Moles et al., 2005; Fan et al., 2006; Orsi and Tanksley, 2009; Gegas et al., 2010). Although the size of seeds is one of the most important agronomic traits in plants, the genetic and molecular mechanisms that set the final size of seeds are almost unknown. In higher plants, seed development starts with a double fertilization process, in which one of the two haploid pollen nuclei fuses with the haploid egg cell to produce the diploid embryo, while the other sperm nucleus fuses with the diploid central cell to form the triploid endosperm (Lopes and Larkins, 1993). The integuments surrounding the ovule are maternal tissues and form the seed coat after fertilization. Therefore, the size of the seed is the result of the growth of the embryo, the endosperm and the maternal tissues. However, the genetic and molecular mechanisms setting the limits of seed growth are almost unknown in plants.
Several factors that function maternally to regulate seed size have been identified in Arabidopsis. For example, TRANSPARENT TESTA GLABRA 2 (TTG2) influences seed growth by increasing cell elongation in the maternal integuments (Garcia et al., 2005; Ohto et al., 2009), while APETALA2 (AP2) may control seed growth by limiting cell elongation in the maternal integuments (Jofuku et al., 2005; Ohto et al., 2005; Ohto et al., 2009). By contrast, AUXIN RESPONSE FACTOR 2 (ARF2) acts maternally to control seed growth by restricting cell proliferation (Schruff et al., 2006). Similarly, the ubiquitin receptor DA1 acts synergistically with the E3 ubiquitin ligases DA2 and EOD1/BB to control seed size by limiting cell proliferation in the maternal integuments (Li et al., 2008; Xia et al., 2013). Mutations in the suppressor of da1-1 (SOD2), which encodes the ubiquitin-specific protease (UBP15), suppress the large seed phenotype of da1-1 (Du et al., 2014). DA1 physically associates with UBP15/SOD2 and modulates the stability of UBP15. These studies show that the ubiquitin pathway plays an important part in the maternal control of seed size. KLU/CYTOCHROME P450 78A5 (CYP78A5) regulates seed size by increasing cell proliferation in the maternal integuments of ovules (Adamski et al., 2009). KLU has also been suggested to generate mobile plant-growth substances that promote cell proliferation (Anastasiou et al., 2007; Adamski et al., 2009). By contrast, overexpression of CYP78A6/EOD3 increases both cell proliferation and cell elongation in the integuments, resulting in large seeds (Fang et al., 2012). Seed size is also determined by zygotic tissues. Several factors have been described to influence seed size via the zygotic tissues in Arabidopsis, including HAIKU1(IKU1), IKU2, MINISEED3 (MINI3) and SHORT HYPOCOTYL UNDER BLUE1 (SHB1) (Garcia et al., 2003; Luo et al., 2005; Zhou et al., 2009; Wang et al., 2010; Kang et al., 2013). iku and mini3 mutants form small seeds due to precocious cellularization of the endosperm (Garcia et al., 2003; Luo et al., 2005; Wang et al., 2010). SHB1 associates with MINI3 and IKU2 promoters and regulates expression of MINI3 and IKU2 (Zhou et al., 2009; Kang et al., 2013). ABA INSENSITIVE5 (AB15) has been recently described to repress the expression of SHB1 (Cheng et al., 2014), and MINI3 has been reported to activate expression of the cytokinin oxidase (CKX2) (Li et al., 2013), suggesting the roles of phytohormones in regulating endosperm growth. In addition, the endosperm growth is influenced by parent of-origin effects (Scott et al., 1998; Xiao et al., 2006).
The invention is aimed at providing plants with improved yield traits that are beneficial to agriculture.
SUMMARY OF THE INVENTIONIn a first aspect, the invention relates to a plant generated that does not produce a functional NGAL2 polypeptide or does not produce functional NGAL2 and NGAL3 polypeptides.
In another aspect, the invention relates to a method for altering a plant phenotype comprising reducing or abolishing the expression of a nucleic acid sequence encoding a NGAL2 polypeptide or reducing or abolishing the activity of a NGAL2 or reducing or abolishing the expression of a nucleic acid sequences encoding NGAL2 and NGAL3 polypeptides or reducing or abolishing the activity of a NGAL2 and NGAL3 polypeptide relative to a control plant.
In another aspect, the invention relates to a method for making a plant with an altered phenotype comprising reducing or abolishing the expression of a nucleic acid sequence encoding a NGAL2 polypeptide or reducing or abolishing the activity of a NGAL2 or reducing or abolishing the expression of a nucleic acid sequences encoding NGAL2 and NGAL3 polypeptides or reducing or abolishing the activity of a NGAL2 and NGAL3 polypeptide relative to a control plant.
In another aspect, the invention relates to a plant obtained or obtainable any method described above.
In another aspect, the invention relates to an isolated nucleic acid comprising a sequence comprising or consisting of SEQ ID NO: 1 or 2 or a functional variant or homologue thereof.
In another aspect, the invention relates to a vector comprising an isolated nucleic acid described above.
In another aspect, the invention relates to a silencing nucleic acid construct targeting sequence comprising or consisting of
1, 2 or 3 or a functional variant, part or homologue thereof.
The invention is further described in the following non-limiting figures.
(A) Seeds from wild-type, da1-1 and sod7-1D da1-1 plants (from left to right). (B) Mature embryos of the wild type, da1-1 and sod7-1D da1-1 (from left to right). (C) Flowers from wild-type, da1-1 and sod7-1D da1-1 plants (from left to right). (D) 30-day-old plants of the wild type, da1-1 and sod7-1D da1-1 (from left to right). (E) Projective area of wild-type, da1-1 and sod7-1D da1-1 seeds. (F) Weight of wild-type, da1-1 and sod7-1D da1-1 seeds. (G) Cotyledon area of 10-d-old wild-type, da1-1 and sod7-1D da1-1 seedlings. Values (E-G) are given as mean±SD relative to the respective wild-type values, set at 100%. **, P<0.01 compared with da1-1 (Student's t-test). Bars=0.5 mm in (A), 0.2 mm in (B), 1 mm in (C) and 5 cm in (D).
(A and B) Seeds of Col-0 (A) and sod7-1D (B). (C and D) Mature embryos of Col-0 (C) and sod7-1D (D). (E and F) 10-day-old seedlings of Col-0 (E) and sod7-1D (F). (G) Projective area of Col-0 and sod7-1D seeds. (H) Weight of Col-0 and sod7-1D seeds. (I) Cotyledon area of 10-day-old Col-0 and sod7-1D seedlings. Values (G-I) are given as mean±SD relative to the respective wild-type values, set at 100%. **, P<0.01 compared with the wild type (Student's t-test). Bars=0.5 mm in (A) and (B), 0.2 mm in (C) and (D), and 1 mm in (E) and (F).
(A) Structure of the T-DNA insertion in the sod7-1D mutant. (B) Expression levels of At3g11580 (SOD7) and At3g11590 in da1-1 and sod7-1D da1 seedlings.
(C) The SOD7 protein contains a B3 DNA binding domain (second domain in lighter shading) and a transcriptional repression motif (small light box in darker shading, marked with an arrow). (D) Projective area of Col-0, 35S:GFP-SOD7#3 and 35S:GFP-SOD7#5 seeds. (E) Cotyledon area of 10-day-old Col-0, 35S:GFP-SOD7#3 and 35S:GFP-SOD7#5 seedlings. (F) Expression levels of SOD7 in Col-0, 35S:GFP-SOD7#3 and 35S:GFP-SOD7#5 seedlings. Values (D-F) are given as mean±SD relative to the respective wild-type values, set at 100%. **, P<0.01 compared with the wild type (Student's t-test).
(A-K) SOD7 expression activity was monitored by pSOD7:GUS transgene expression. Histochemical analysis of GUS activity in the developing leaves (A, B and C), the developing sepals (D, E), the developing petals (F, G), the developing stamens (H, I), and the developing carpels (J, K). (L) GFP florescence of SOD7-GFP in a young ovule of pSOD7:SOD7-GFP transgenic plants. (M-O) GFP fluorescence of SOD7-GFP (M), DAPI staining (N), and merged (0) images are shown. Epidermal cells in pSOD7:SOD7-GFP leaves were used to observe GFP signal. (P-R) GFP fluorescence of GFP-SOD7 (P), DAPI staining (Q), and merged (R) images are shown. Epidermal cells in 35S:GFP-SOD7 leaves were used to observe GFP signal. Bars=100 μm in (A-K), 10 μm in (L), and 2 μm in (M-R).
(A) The SOD7 gene structure. The start codon (ATG) and the stop codon (TGA) are shown. Closed boxes indicate the coding sequence, and the line between boxes indicates intron. The T-DNA insertion site (sod7-ko1) in the SOD7 gene was indicated.
(B) The NGAL3 gene structure. The start codon (ATG) and the stop codon (TGA) are shown. Closed boxes indicate the coding sequence, and the line between boxes indicates intron. The T-DNA insertion site (ngal3-ko1) in the NGAL3 gene was indicated. (C) Seeds from Col-0, sod7-ko1, ngal3-ko1 and sod7-ko1 ngal3-ko1 plants (from left to right). (D) Mature embryos of Col-0, sod7-ko1, ngal3-ko1 and sod7-ko1 ngal3-ko1 (from left to right). (E) 25-day-old plants of Col-0, sod7-ko1, ngal3-ko1 and sod7-ko1 ngal3-ko1 (from left to right). (F) Flowers of Col-0, sod7-ko1, ngal3-ko1 and sod7-ko1 ngal3-ko1 (from left to right). (G) Projective area of Col-0, sod7-ko1, ngal3-ko1 and sod7-ko1 ngal3-ko1 seeds. (H) Weight of Col-0, sod7-ko1, ngal3-ko1 and sod7-ko1 ngal3-ko1 seeds. (I) Cotyledon area of Col-0, sod7-ko1, ngal3-ko1 and sod7-ko1 ngal3-ko1 seedlings. Values (G-I) are given as mean±SD relative to the respective wild-type values, set at 100%. **, P<0.01 compared with the wild type (Col-0) (Student's t-test). Bars=0.5 mm in (C), 0.2 mm in (D), 5 cm in (E), and 1 mm in (F).
(A) Projective area of Col-0×Col-0 (C/C) F1, Col-0×sod7-ko1 ngal3-ko1 (C/d) F1, sod7-ko1 ngal3-ko1×Col-0 (d/C) F1 and sod7-ko1 ngal3-ko1×sod7-ko1 ngal3-ko1 (d/d) F1 seeds. Values are given as mean±SD relative to the respective wild-type values, set at 100%. (B) Projective area of Col-0×Col-0 (C/C) F2, Col-0×sod7-ko1 ngal3-ko1 (C/d) F2, sod7-ko1 ngal3-ko1×Col-0 (d/C) F2 and sod7-ko1 ngal3-ko1×sod7-ko1 ngal3-ko1 (d/d) F2 seeds. Values are given as mean±SD relative to the respective wild-type values, set at 100%. (C and D) Mature ovules of Col-0 (C) and sod7-ko1 ngal3-ko1 (D). (E) Outer integument length of mature Col-0 (lighter bar to the left) and sod7-ko1 ngal3-ko1 (darker bar to the right) ovules. Values are given as mean±SD. (F) The number of cells in the outer integuments of Col-0 and sod7-ko1 ngal3-ko1 at 0, 6 and 8 DAP. Values are given as mean±SD. (F) The length of cells in the outer integuments of Col-0 and sod7-ko1 ngal3-ko1 at 0, 6 and 8 DAP. Values are given as mean±SD. **, P<0.01 compared with the wild type (Col-0) (Student's t-test). Bars=50 μm in (C) and (D).
(A) Seed area of Col-0, klu-4, sod7-ko1 ngal3-ko1 and klu-4 sod7-ko1 ngal3-ko1 (from left to right). Values are given as mean±SD relative to the respective wild-type values, set at 100%. (B) Seed weight of Col-0, klu-4, sod7-ko1 ngal3-ko1 and klu-4 sod7-ko1 ngal3-ko1 (from left to right). Values are given as mean±SD relative to the respective wild-type values, set at 100%. (C) The outer integument length of Col-0, klu-4, sod7-ko1 ngal3-ko1 and klu-4 sod7-ko1 ngal3-ko1 (from left to right). ngal3-ko1 at 0 and 8 DAP. Values are given as mean±SD. (D) The number of cells in the outer integuments of Col-0, klu-4, sod7-ko1 ngal3-ko1 and klu-4 sod7-ko1 ngal3-ko1 (from left to right) at 0 and 8 DAP. Values are given as mean±SD. **, P<0.01 compared with their respective controls (Student's t-test).
(A) Expression dynamics of SOD7 and KLU in pER8-SOD7 transgenic plants treated with β-estradiol for 0, 4 and 8 hours. Means were calculated from three biological samples. Values are given as mean±SD. **, P<0.01, compared with the expression level of KLU and SOD7 at 0 hour, respectively (Student's t-test). (B) A 2-kb promoter region of KLU upstream of its ATG codon contains a CACTTG sequence. PF1 and PF2 represent PCR fragments used for ChIP-quantitative PCR analysis. A and A-m indicate the wild-type probe and the mutated probe used in the EMSA essay, respectively. (C) ChIP-qPCR analysis shows that SOD7 binds to the promoter fragment PF1 of KLU. Chromatin from 35S:GFP and 35S:GFP-SOD7 transgenic plants was immunoprecipitated by anti-GFP, and the enrichment of the fragments was determined by quantitative real-time PCR. The ACTIN7 promoter was used as a negative control. The fold enrichment was normalized to the ACTIN7 amplicon, set at 1. Means were calculated from three biological samples. Values are given as mean±SD. **, P<0.01, compared with 35S:GFP transgenic plants (Student's t-test). (D) Direct interaction between SOD7 and the KLU promoter determined by EMSA. The biotin-labeled probe A and MBP-SOD7 formed the DNA-protein complex, but the mutated probe A-m and MBP-SOD7 did not form the DNA-protein complex. The retarded DNA-protein complex was reduced by competition using the unlabeled probe A.
Overexpression of SOD7 results in small plants compared with the wild type. Bar=5 cm.
Petal area of Col-0, sod7-ko1, ngal3-ko1 and sod7-ko1 ngal3-ko1. (B) The seventh leaf area of Col-0, sod7-ko1, ngal3-ko1 and sod7-ko1 ngal3-ko1. Values (A and B) are given as mean±SD relative to the respective wild-type values, set at 100%. **, P<0.01 and *, P<0.05 compared with the wild type (Col-0).
The present invention will now be further described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of botany, microbiology, tissue culture, molecular biology, chemistry, biochemistry and recombinant DNA technology, bioinformatics which are within the skill of the art. Such techniques are explained fully in the literature.
As used herein, the words “nucleic acid”, “nucleic acid sequence”, “nucleotide”, “nucleic acid molecule” or “polynucleotide” are intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), naturally occurring, mutated, synthetic DNA or RNA molecules, and analogues of the DNA or RNA generated using nucleotide analogues. It can be single-stranded or double-stranded. Such nucleic acids or polynucleotides include, but are not limited to, coding sequences of structural genes, anti-sense sequences, and non-coding regulatory sequences that do not encode mRNAs or protein products. These terms also encompass a gene. The term “gene” or “gene sequence” is used broadly to refer to a DNA nucleic acid associated with a biological function. Thus, genes may include introns and exons as in the genomic sequence, or may comprise only a coding sequence as in cDNAs, and/or may include cDNAs in combination with regulatory sequences.
The terms “peptide”, “polypeptide” and “protein” are used interchangeably herein and refer to amino acids in a polymeric form of any length, linked together by peptide bonds.
For 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) both (a) and (b)
are not located in their natural genetic environment or have been modified by genetic intervention techniques, 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 both incorporated by reference.
In certain embodiments, 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. Thus, the plant can express a silencing construct transgene. However, as mentioned, in certain embodiments, transgenic also means that, while the nucleic acids according to the different embodiments of the invention 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, for example by mutagenesis.
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. According to the invention, the transgene is stably integrated into the plant and the plant is preferably homozygous for the transgene.
The various aspects of the invention use genetic engineering methods. Thus, the plants have been generated using genetic engineering methods, for example transgene expression, mutagenesis, gene targeting, gene silencing or genome editing as detailed below. Thus, the various aspects of the invention can involve recombinant DNA technology. The plants of the invention are thus mutant plants which have been genetically engineered, that is manipulated by human intervention. The plants of the various aspects of the invention do not relate to natural variants which have not been manipulated by genetic engineering methods. The plant may be a transgenic plant in some embodiments, for example a plant which comprises a nucleic acid construct expressing a silencing construct.
In preferred embodiments exclude embodiments that are solely based on generating plants by traditional breeding methods.
The inventor has identified a B3 domain transcriptional repressor termed AtNGAL2, encoded by the suppressor of Atda1-1 (AtSOD7), which acts maternally to control seed size by restricting cell proliferation in the integuments of ovules and developing seeds.
The inventor previously identified the ubiquitin receptor DA1 as a negative regulator of seed size in Arabidopsis (Li et al., 2008). The da1-1 mutant formed large seeds due to increased cell proliferation in the maternal integuments (Li et al., 2008; Xia et al., 2013). To identify novel components in the DA1 pathway or other seed size regulators, the inventor initiated a T-DNA activation tagging screen for modifiers of da1-1 (Fang et al., 2012). A dominant suppressor of da1-1 (sod7-1D) was isolated from seeds produced from approximate 16,000 T1 plants (
The inventor has shown that overexpression of AtSOD7 significantly decreases seed size of wild-type plants, while the disruption of AtSOD7 increases seed size. The inventors have shown that disruption of AtNGAL3, a close homolog of AtSOD7 also increases seed size. Moreover, the simultaneous disruption of AtSOD7 and AtNGAL3 further increases seed size in a synergistic manner. Genetic analyses carried out by the inventor indicate that AtSOD7 acts in a common pathway with the seed size regulator AtKLU to control seed growth, but does so independently of AtDA1. Further results show that AtSOD7 directly binds to the promoter of AtKLU in vitro and in vivo and represses expression of AtKLU. Therefore, the inventor's findings show that AtSOD7 (aka AtNGAL2) is a target for seed size improvement in crops. The plants of the invention are characterised by increased organ size, for example increased seed size, and also increased petal size, increased embryo size, for example. Increased seed size leads to an increase in seed yield and the plants of the invention are thus characterised by increased seed yield.
Thus, the invention relates to a plant wherein said plant does not produce a functional NGAL2 and/or NGAL3 polypeptide. For example, the plant does not produce a full length transcript of a nucleic acid sequence encoding a NGAL2 and/or NGAL3 protein. In another embodiment, the plant produces a full length transcript of a nucleic acid sequence encoding a NGAL2 and/or NGAL3, but the resulting protein is not functional. In a preferred embodiment, said plant does not produce a functional NGAL2 polypeptide and also does not produce a functional NGAL3 polypeptide. Such plants are double knock-out or knock-down mutants (loss of function mutants) and methods according to the invention as described below relate to making such double mutants.
The plants of the invention are mutant plants which have been genetically modified and are not naturally occurring varieties. Thus, the plants have been generated using genetic engineering methods, for example mutagenesis, gene targeting, gene silencing or genome editing as detailed below. Thus, the various aspects of the invention can involve recombinant DNA technology. The plant may be a transgenic plant in some embodiments, for example a plant which comprises a transgene to silence gene expression of SOD7 and/or NGAL3. In other embodiments, the plant does not carry a transgene, but is a mutant plant wherein the endogenous nucleic acid sequence encoding a NGAL2 and/or NGAL3 polypeptide or the endogenous SOD7 and/or NGAL3 promoter sequence has been manipulated to either reduce or abolish expression of a nucleic acid sequence encoding a NGAL2 and/or NGAL3 polypeptide or reduce or abolish the activity of a NGAL2 and/or NGAL3 polypeptide. The plants of the various aspects of the invention do not relate to natural variants which have not been manipulated by genetic engineering methods.
In one aspect, the invention relates to a plant generated by genetic engineering methods wherein the expression of a nucleic acid sequence encoding a NGAL2 and/or NGAL3 polypeptide and/or the activity of a NGAL2 and/or NGAL3 polypeptide is reduced or abolished relative to a control plant. In one embodiment, expression of a nucleic acid sequence encoding a NGAL2 polypeptide or the activity of a NGAL2 polypeptide is reduced or abolished. In another embodiment, expression of a nucleic acid sequence encoding a NGAL3 polypeptide or the activity of a NGAL3 polypeptide is reduced or abolished. In a preferred embodiment the presence of function of both proteins is affected, in other words, the plant is characterised in that expression of a nucleic acid sequence encoding a NGAL2 polypeptide or the activity of a NGAL2 polypeptide is reduced or abolished and also expression of a nucleic acid sequence encoding a NGAL3 polypeptide or the activity of a NGAL3 polypeptide is reduced or abolished in said plant.
For example, said plant can have reduced or abolished expression of a nucleic acid sequence encoding a NGAL2 polypeptide and reduced or abolished expression of a nucleic acid sequence encoding a NGAL3 polypeptide. In another embodiment, said plant can have reduced or abolished activity of a NGAL2 polypeptide and reduced or abolished activity of a NGAL3 polypeptide. In another embodiment, said plant can have reduced or abolished expression of a nucleic acid sequence encoding a NGAL2 polypeptide and reduced or abolished activity of a NGAL3 polypeptide. In another embodiment, said plant can have reduced or abolished expression of a nucleic acid sequence encoding a NGAL3 polypeptide and reduced or abolished activity of a NGAL2 polypeptide.
A NGAL2 or NGAL3 polypeptide as described in the various aspects of the invention has a characteristic domain structure as explained below.
A NGAL2 OR NGLA3 polypeptide as described in the various aspects of the invention comprises a B3 DNA binding domain which has the structure shown in
In one embodiment, the domain is: SNNNNNNGGSGDDVACHFQRFDLHRLFIGWRGE (SEQ ID NO:6) or a domain with at least 80%, at least 95% or at least 95% sequence identity thereto.
A NGAL2 OR NGAL3 polypeptide as described in the various aspects of the invention also comprises a transcriptional repression motif shown in
In one embodiment, the domain is: VRLFGVNLE (SEQ ID NO:7) or a domain with at least 95% sequence identity thereto.
In one embodiment, the NGAL2 protein is AtNGAL2, a functional variant, part or homologue thereof. AtNGAL2 is encoded by AtSOD7. The term AtSOD7 refers to the wild type AtSOD7 nucleic acid sequence comprising or consisting of SEQ ID NO. 1 (CDNA) or SEQ ID NO 2 (genomic DNA). The protein encoded by AtSOD7 is termed AtNGAL2 SEQ ID NO.3. In one embodiment, said functional homologue is not AtNGAL3.
In one embodiment, the NGAL3 protein is AtNGAL3, a functional variant, part or homologue thereof. The term AtNGAL3 refers to the wild type AtNGAL3 nucleic acid sequence comprising or consisting of SEQ ID NO. 4. The protein encoded by AtNGAL3 is termed AtNGAL3 SEQ ID NO.5.
The term “functional” refers to the biological function of the NGAL2 or NGAL3, that is their function in controlling organ size, in particular seed size. The terms “functional variant” or “functional part” as used herein, for example with reference to SEQ ID NOs: 1, 2 or 3, or SEQ ID NOs: 4 or 5 refers to a variant gene or polypeptide sequence or part of the gene or polypeptide sequence which retains the biological function of the full non-variant SOD7/NGAL2 or NGAL2/NGAL3 sequence, that is regulation of seed size. Such sequences complement the Atsod7-1D mutant or Atngal3 mutant respectively.
Thus, it is understood, as those skilled in the art will appreciate, that the aspects of the invention, encompass not only targeting a AtSOD7 and/or AtNGAL3 nucleic acid, for example a nucleic acid sequence comprising or consisting of SEQ ID NO: 1 or SEQ ID NO: 2, or SEQ ID NO: 4 respectively or a polypeptide comprising or consisting of SEQ ID NO: 3, or SEQ ID NO: 5, or a promoter of a AtSOD7 and/or AtNGAL3 nucleic acid. The aspects of the invention encompass also functional variants of AtNGAL2 or AtNGAL3 that do not affect the biological activity and function of the resulting protein. Alterations in a nucleic acid sequence which result in the production of a different amino acid at a given site that do however not affect the functional properties of the encoded polypeptide, are well known in the art. For example, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also produce a functionally equivalent product. Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products. Also encompassed is a variant that is substantially identical, i.e. has only some sequence variations, for example in non-conserved residues, to the wild type sequences as shown herein and is biologically active.
Generally, variants of a particular SOD7/NGAL3 nucleotide sequence or NGAL2/NGAL3 polypeptide as described herein will have at least about 60%, preferably at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to that particular non-variant nucleotide sequence, as determined by sequence alignment programs described elsewhere herein.
Furthermore, the various the aspects of the invention encompass not only a AtSOD7 and/or AtNGAL3 nucleic acid, for example a nucleic acid sequence comprising or consisting of
1 or SEQ ID NO: 2, or SEQ ID NO: 4 respectively or a polypeptide comprising or consisting of SEQ ID NO: 3, or SEQ ID NO: 5, or their functional variants but also homologues of AtSOD7 and/or AtNGAL3 in Arabidopsis or other plants. Also within the scope of the invention are functional variants of such homologues as defined above.
The term homologue as used herein also designates an AtSOD7 and/or AtNGAL3 orthologue from other plant species. A homologue of AtNGAL2 or AtNGAL3 polypeptide respectively has, in increasing order of preference, at least 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 at least 99% overall sequence identity to the amino acid represented by SEQ ID NO: 3 or 5 respectively. Preferably, overall sequence identity is at least 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%, most preferably 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%.
In another embodiment, the homologue of a AtSOD7 or AtNGAL3 nucleic acid sequence respectively has, in increasing order of preference, at least 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 at least 99% overall sequence identity to the nucleic acid represented by SEQ ID NO: 1 or 2 or 4 respectively.
Preferably, overall sequence identity is at least 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%, most preferably 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%. The overall sequence identity is determined using a global alignment algorithm known in the art, such as the Needleman Wunsch algorithm in the program GAP (GCG Wisconsin Package, Accelrys).
In a preferred embodiment, the NGAL2 or NGAL3 homologue is from a plant that is not Arabidopsis.
In one embodiment, an AtNGAL2 or a homologue thereof or AtNGAL3 or a homologue thereof comprises a B3 domain having the sequence as defined above
In one embodiment, an AtNGAL2 or a homologue thereof or AtNGAL3 or a homologue thereof comprises a transcriptional repression motif having the sequence as defined above
Examples of homologues are shown in
Thus, the nucleotide sequences of the invention and described herein can also be used to isolate corresponding sequences from other organisms, particularly other plants, for example crop plants. In this manner, methods such as PCR, hybridization, and the like can be used to identify such sequences based on their sequence homology to the sequences described herein. Topology of the sequences and the characteristic domains structure can also be considered when identifying and isolating homologues. Sequences may be isolated based on their sequence identity to the entire sequence or to fragments thereof. In hybridization techniques, all or part of a known nucleotide sequence is used as a probe that selectively hybridizes to other corresponding nucleotide sequences present in a population of cloned genomic DNA fragments or cDNA fragments (i.e., genomic or cDNA libraries) from a chosen plant. The hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labelled with a detectable group, or any other detectable marker. Thus, for example, probes for hybridization can be made by labelling synthetic oligonucleotides based on the ABA-associated sequences of the invention. Methods for preparation of probes for hybridization and for construction of cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook, et al., (1989) Molecular Cloning: A Library Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).
Hybridization of such sequences may be carried out under stringent conditions. By “stringent conditions” or “stringent hybridization conditions” is intended conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences that are 100% complementary to the probe can be identified (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about 1000 nucleotides in length, preferably less than 500 nucleotides in length.
Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Duration of hybridization is generally less than about 24 hours, usually about 4 to 12. Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
According to the invention, preferred homologues of AtSOD7 and AtNGAL3 peptides are selected from crop plants, for example cereal crops. Preferred homologues of AtNGAL2 and AtNGAL3 and their polypeptide sequences are also shown in
A plant according to the various aspects of the invention, including the transgenic plants, methods and uses described herein may be a monocot or a dicot plant.
A dicot plant may be selected from the families including, but not limited to Asteraceae, Brassicaceae (e.g. Brassica napus), Chenopodiaceae, Cucurbitaceae, Leguminosae (Caesalpiniaceae, Aesalpiniaceae Mimosaceae, Papilionaceae or Fabaceae), Malvaceae, Rosaceae or Solanaceae. For example, the plant may be selected from lettuce, sunflower, Arabidopsis, broccoli, spinach, water melon, squash, cabbage, tomato, potato, yam, capsicum, tobacco, cotton, okra, apple, rose, strawberry, alfalfa, bean, soybean, field (fava) bean, pea, lentil, peanut, chickpea, apricots, pears, peach, grape vine, bell pepper, chilli or citrus species.
A monocot plant may, for example, be selected from the families Arecaceae, Amaryllidaceae or Poaceae. For example, the plant may be a cereal crop, such as maize, wheat, rice, barley, oat, sorghum, rye, millet, buckwheat, or a grass crop such as Lolium species or Festuca species, or a crop such as sugar cane, onion, leek, yam or banana.
Also included are biofuel and bioenergy crops such as rape/canola, sugar cane, sweet sorghum, Panicum virgatum (switchgrass), linseed, lupin and willow, poplar, poplar hybrids, Miscanthus or gymnosperms, such as loblolly pine. Also included are crops for silage (maize), grazing or fodder (grasses, clover, sanfoin, alfalfa), fibres (e.g. cotton, flax), building materials (e.g. pine, oak), pulping (e.g. poplar), feeder stocks for the chemical industry (e.g. high erucic acid oil seed rape, linseed) and for amenity purposes (e.g. turf grasses for golf courses), ornamentals for public and private gardens (e.g. snapdragon, petunia, roses, geranium, Nicotiana sp.) and plants and cut flowers for the home (African violets, Begonias, chrysanthemums, geraniums, Coleus spider plants, Dracaena, rubber plant).
Preferably, the plant is a crop plant. By crop plant is meant any plant which is grown on a commercial scale for human or animal consumption or use. In a preferred embodiment, the plant is a cereal.
Most preferred plants are maize, rice, wheat, oilseed rape/canola, sorghum, soybean, sunflower, alfalfa, potato, tomato, tobacco, grape, barley, pea, bean, field bean, lettuce, cotton, sugar cane, sugar beet, broccoli or other vegetable brassicas or poplar.
The term “plant” as used herein encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, fruit, 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.
According to the various aspects of the invention, including the plants and methods of the invention, abolishing, inactivating, repressing, reducing or down-regulating the activity of a NGAL2 and/or NGAL3 polypeptide can be achieved through different means. Such means that are within the scope of the various aspects of the invention are methods for abolishing or reducing translation or transcription of the SOD7 and/or NGAL3 gene, destabilizing SOD7 and/or NGAL3 transcript stability, destabilizing NGAL2 and/or NGAL3 polypeptide stability or abolishing or reducing the activation or activity of the NGAL2 and/or NGAL3 or polypeptide. Thus, in one embodiment, endogenous SOD7 and/or NGAL3 gene or its promoter carry a functional mutation so that no full length transcript is made. In another embodiment, the SOD7 and/or NGAL3 gene is silenced in said plant using gene silencing techniques. In another embodiment, the SOD7 and/or NGAL3 nucleic acid sequence has been altered to introduce a mutation which results in a NGAL2/NGAL3 protein with reduced or abolished activity. These embodiments and the techniques used are described in more detail below.
In another aspect, the invention relates to a method for altering a plant phenotype comprising reducing or abolishing the expression of a nucleic acid sequence encoding a NGAL2 and/or NGAL3 polypeptide and/or reducing or abolishing the activity of a NGAL2 and/or NGAL3 polypeptide relative to a control plant.
In another aspect, the invention relates to a method for making a plant with an altered phenotype comprising reducing or abolishing the expression of a nucleic acid sequence encoding a NGAL2 and/or NGAL3 polypeptide and/or reducing or abolishing the activity of a NGAL2 and/or NGAL3 polypeptide relative to a control plant.
As previously described, such methods above use genetic engineering methods.
In this aspect, a wild type plant may be targeted to simultaneously knock out or down both SOD7 and NGAL3 function. Alternatively, the method may comprise the following steps
-
- a) Knocking out or down SOD7 function in a first plant;
- b) knocking out or down NGAL3 function in a second plant and
- c) crossing plants regenerated from said first plant with plants regenerated from said second plant.
In one embodiment of these methods, expression of a nucleic acid sequence encoding a NGAL2 polypeptide or the activity of a NGAL2 polypeptide is reduced or abolished. In another embodiment, expression of a nucleic acid sequence encoding a NGAL3 polypeptide or the activity of a NGAL3 polypeptide is reduced or abolished. In a preferred embodiment, the method comprises reducing or abolishing expression of a nucleic acid sequence encoding a NGAL2 polypeptide or the activity of a NGAL2 polypeptide and reducing or abolishing expression of a nucleic acid sequence encoding a NGAL3 polypeptide or the activity of a NGAL3 polypeptide to create a double loss of function mutant.
For example, the method comprises reducing or abolishing expression of a nucleic acid sequence encoding a NGAL2 polypeptide and reducing or abolishing expression of a nucleic acid sequence encoding a NGAL3 polypeptide. In another embodiment, the method comprises reducing or abolishing activity of a NGAL2 polypeptide and reducing or abolishing activity of a NGAL3 polypeptide. In another embodiment, the method comprises reducing or abolishing expression of a nucleic acid sequence encoding a NGAL2 polypeptide and reducing or abolishing activity of a NGAL3 polypeptide. In another embodiment the method comprises reducing or abolishing expression of a nucleic acid sequence encoding a NGAL3 polypeptide or reducing or abolishing activity of a NGAL2 polypeptide.
According to these methods, the phenotype is preferably selected from increased organ size, for example increased seed size or increased seed weight. Increased seed size leads to an increase in yield and the methods of the invention also increased yield.
The 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” as described herein relates to yield-related traits and may relate to vegetative biomass (root and/or shoot biomass), to reproductive organs, and/or to propagules (such as seeds) of that plant. Thus, according to the invention, the term yield refers to organ size, in particular seed size and can be measured by assessing seed size or seed weight or cotyledon size.
The terms “increase”, “improve” or “enhance” are interchangeable. Yield or seed size for example is increased by at least a 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%, preferably at least 15% or 20%, more preferably 25%, 30%, 35%, 40% or 50% or more in comparison to a control plant.
A control plant as used herein according to all of the aspects of the invention is a plant which has not been modified according to the methods of the invention. Accordingly, the control plant has not been genetically modified to alter either expression of a nucleic acid encoding a NGAL2 or NGAL3 polypeptide or to alter the activity of a NGAL2 or NGAL3 polypeptide as described herein. In one embodiment, the control plant is a wild type plant that has not been genetically altered. In another embodiment, the control plant is a transgenic plant that does not have altered expression of a nucleic acid encoding a NGAL2 or NGAL3 polypeptide or altered activity of a NGAL2 or NGAL3 polypeptide, but has been genetically altered in other ways, for example by expressing a desirable transgene to confer certain traits.
The reduction, decrease, down-regulation or repression of the activity of the NGAL2 and/or NGAL3 polypeptide or corresponding SOD7 and/or NGAL3 nucleic acid sequences according to the aspects of the invention is at least 10%, 20%, 30%, 40% or 50% in comparison to the control plant.
For example, the plant is a reduction (knock down) or loss of function (knock out) mutant wherein the function of the SOD7 and/or NGAL3 nucleic acid sequence is reduced or lost compared to a wild type control plant. To this end, a mutation is introduced into the SOD7 and/or NGAL3 nucleic acid sequence or the corresponding promoter sequence which disrupts the transcription of the gene leading to a gene product which is not functional or has a reduced function. The mutation may be a deletion, insertion or substitution. The expression of active protein may thus be abolished by mutating the nucleic acid sequences in the plant cell which encode the NGAL2 or NGAL3 polypeptide and regenerating a plant from the mutated cell. The nucleic acids may be mutated by insertion or deletion of one or more nucleotides. Techniques for the inactivation or knockout of target genes are well-known in the art. These techniques include gene target using vectors that target the gene of interest and which allow integration allows for integration of transgene at a specific site. The targeting construct is engineered to recombine with the target gene, which is accomplished by incorporating sequences from the gene itself into the construct. Recombination then occurs in the region of that sequence within the gene, resulting in the insertion of a foreign sequence to disrupt the gene. With its sequence interrupted, the altered gene will be translated into a nonfunctional protein, if it is translated at all. Other techniques include genome editing (targeted genome engineering) as described below. Using either of these techniques, in preferred embodiment, conserved domains which confer function of NGAL2 or NGAL3 respectively are modified.
A skilled person will know further approaches can be used to generate such mutants. In one embodiment, insertional mutagenesis is used, for example using T-DNA mutagenesis (which inserts pieces of the T-DNA from the Agrobacterium tumefaciens T-Plasmid into DNA causing either loss of gene function or gain of gene function mutations), site-directed nucleases (SDNs) or transposons as mutagens. Insertional mutagenesis is an alternative means of disrupting gene function and is based on the insertion of foreign DNA into the gene of interest (see Krysan et al, The Plant Cell, Vol. 11, 2283-2290, December 1999).
In one embodiment, as discussed in the examples, T-DNA may be used as an insertional mutagen which disrupts SOD7 and/or NGAL3 gene expression. T-DNA not only disrupts the expression of the gene into which it is inserted, but also acts as a marker for subsequent identification of the mutation. Since the sequence of the inserted element is known, the gene in which the insertion has occurred can be recovered, using various cloning or PCR-based strategies. The insertion of a piece of T-DNA on the order of 5 to 25 kb in length generally produces a disruption of gene function. If a large enough population of T-DNA transformed lines is generated, there are reasonably good chances of finding a transgenic plant carrying a T-DNA insert within any gene of interest. Transformation of spores with T-DNA is achieved by an Agrobacterium-mediated method which involves exposing plant cells and tissues to a suspension of Agrobacterium cells.
The details of this method are well known to a skilled person. In short, plant transformation by Agrobacterium results in the integration into the nuclear genome of a sequence called T-DNA, which is carried on a bacterial plasmid. The use of T-DNA transformation leads to stable single insertions. Further mutant analysis of the resultant transformed lines is straightforward and each individual insertion line can be rapidly characterized by direct sequencing and analysis of DNA flanking the insertion. Gene expression in the mutant is compared to expression of the SOD7 and/or NGAL3 nucleic acid sequence in a wild type plant and phenotypic analysis is also carried out. Other techniques for insertional mutagenesis include the use of transposons.
In another embodiment, mutagenesis is physical mutagenesis, such as application of ultraviolet radiation, X-rays, gamma rays, fast or thermal neutrons or protons. The targeted population can then be screened to identify a SOD7 or NGAL3 loss of function mutant.
In another embodiment of the various aspects of the invention, the plant is a mutant plant derived from a plant population mutagenised with a mutagen. The mutagen may be fast neutron irradiation or a chemical mutagen, for example selected from the following non-limiting list: ethyl methanesulfonate (EMS), methylmethane sulfonate (MMS), N-ethyl-N-nitrosurea (ENU), triethylmelamine (1′EM), N-methyl-N-nitrosourea (MNU), procarbazine, chlorambucil, cyclophosphamide, diethyl sulfate, acrylamide monomer, melphalan, nitrogen mustard, vincristine, dimethylnitosamine, N-methyl-N′-nitro-Nitrosoguanidine (MNNG), nitrosoguanidine, 2-aminopurine, 7,12 dimethyl-benz(a)anthracene (DMBA), ethylene oxide, hexamethylphosphoramide, bisulfan, diepoxyalkanes (diepoxyoctane (DEO), diepoxybutane (BEB), and the like), 2-methoxy-6-chloro-9 [3-(ethyl-2-chloroethyl)aminopropylamino]acridine dihydrochloride (ICR-170) or formaldehyde.
In one embodiment, the method used to create and analyse mutations is targeting induced local lesions in genomes (TLLING), reviewed in Henikoff et al, 2004. In this method, seeds are mutagenised with a chemical mutagen, for example EMS. The resulting M1 plants are self-fertilised and the M2 generation of individuals is used to prepare DNA samples for mutational screening. DNA samples are pooled and arrayed on microtiter plates and subjected to gene specific PCR. The PCR amplification products may be screened for mutations in the SOD7 and/or NGAL3 target gene using any method that identifies heteroduplexes between wild type and mutant genes. For example, but not limited to, denaturing high pressure liquid chromatography (dHPLC), constant denaturant capillary electrophoresis (CDCE), temperature gradient capillary electrophoresis (TGCE), or by fragmentation using chemical cleavage. Preferably the PCR amplification products are incubated with an endonuclease that preferentially cleaves mismatches in heteroduplexes between wild type and mutant sequences. Cleavage products are electrophoresed using an automated sequencing gel apparatus, and gel images are analyzed with the aid of a standard commercial image-processing program. Any primer specific to the SOD7 or NGAL3 nucleic acid sequence may be utilized to amplify the SOD7 or NGAL3 nucleic acid sequence within the pooled DNA sample. Preferably, the primer is designed to amplify the regions of the SOD7 and/or NGAL3 gene where useful mutations are most likely to arise, specifically in the areas of the SOD7 and/or NGAL3 gene that are highly conserved and/or confer activity as explained elsewhere. To facilitate detection of PCR products on a gel, the PCR primer may be labelled using any conventional labelling method.
Rapid high-throughput screening procedures thus allow the analysis of amplification products for identifying a mutation conferring the reduction or inactivation of the expression of the SOD7 and/or NGAL3 gene as compared to a corresponding non-mutagenised wild type plant. Once a mutation is identified in a gene of interest, the seeds of the M2 plant carrying that mutation are grown into adult M3 plants and screened for the phenotypic characteristics associated with the target gene SOD7 or NGAL3. Loss of function or reduced function mutants with increased seed size compared to a control can thus be identified.
Plants obtained or obtainable by such method which carry a functional mutation in the endogenous SOD7 and/or NGAL3 locus are also within the scope of the invention
In another embodiment, RNA-mediated gene suppression or RNA silencing may be used to achieve silencing of the SOD7 and/or NGAL3 nucleic acid sequence. “Gene silencing” is a term generally used to refer to suppression of expression of a gene via sequence-specific interactions that are mediated by RNA molecules. The degree of reduction may be so as to totally abolish production of the encoded gene product, but more usually the abolition of expression is partial, with some degree of expression remaining. The term should not therefore be taken to require complete “silencing” of expression.
Transgenes may be used to suppress endogenous plant genes. This was discovered originally when chalcone synthase transgenes in petunia caused suppression of the endogenous chalcone synthase genes and indicated by easily visible pigmentation changes. Subsequently it has been described how many, if not all plant genes can be “silenced” by transgenes. Gene silencing requires sequence similarity between the transgene and the gene that becomes silenced. This sequence homology may involve promoter regions or coding regions of the silenced target gene. When coding regions are involved, the transgene able to cause gene silencing may have been constructed with a promoter that would transcribe either the sense or the antisense orientation of the coding sequence RNA. It is likely that the various examples of gene silencing involve different mechanisms that are not well understood. In different examples there may be transcriptional or post-transcriptional gene silencing and both may be used according to the methods of the invention.
The mechanisms of gene silencing and their application in genetic engineering, which were first discovered in plants in the early 1990s and then shown in Caenorhabditis elegans are extensively described in the literature.
RNA-mediated gene suppression or RNA silencing according to the methods of the invention includes co-suppression wherein over-expression of the target sense RNA or mRNA, that is the SOD7 and/or NGAL3 sense RNA or mRNA, leads to a reduction in the level of expression of the genes concerned. RNAs of the transgene and homologous endogenous gene are coordinately suppressed. Other techniques used in the methods of the invention include antisense RNA to reduce transcript levels of the endogenous target gene in a plant. In this method, RNA silencing does not affect the transcription of a gene locus, but only causes sequence-specific degradation of target mRNAs. An “antisense” nucleic acid sequence comprises a nucleotide sequence that is complementary to a “sense” nucleic acid sequence encoding a NGAL2 and/or NGAL3 protein, or a part of the 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 SOD7 and/or NGAL3 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 SOD7 and/or NGAL3 nucleic acid sequence, 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. 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 hybridize with or bind to mRNA transcripts and/or insert into 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 vectors.
RNA interference (RNAi) is another post-transcriptional gene-silencing phenomenon which may be used according to the methods of the invention. This is induced by double-stranded RNA in which mRNA that is homologous to the dsRNA is specifically degraded. It refers to the process of sequence-specific post-transcriptional gene silencing mediated by short interfering RNAs (siRNA). The process of RNAi begins when the enzyme, DICER, encounters dsRNA and chops it into pieces called small-interfering RNAs (siRNA). This enzyme belongs to the RNase III nuclease family. A complex of proteins gathers up these RNA remains and uses their code as a guide to search out and destroy any RNAs in the cell with a matching sequence, such as target mRNA.
Artificial and/or natural microRNAs (miRNAs) may be used to knock out gene expression and/or mRNA translation. MicroRNAs (miRNAs) miRNAs are typically single stranded small RNAs typically 19-24 nucleotides long. Most plant 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 microRNA (amiRNA) technology has been applied in Arabidopsis thaliana and other plants to efficiently silence target genes of interest. The design principles for amiRNAs have been generalized and integrated into a Web-based tool (wmd.weigelworld.org).
Thus, according to the various aspects of the invention a plant may be transformed to introduce a RNAi, shRNA, snRNA, dsRNA, siRNA, miRNA, ta-siRNA, amiRNA or cosuppression molecule that has been designed to target the expression of an SOD7 and/or NGAL3 nucleic acid sequence and selectively decreases or inhibits the expression of the gene or stability of its transcript. Preferably, the RNAi, snRNA, dsRNA, shRNA siRNA, miRNA, amiRNA, to-siRNA or cosuppression molecule used according to the various aspects of the invention comprises a fragment of at least 17 nt, preferably 22 to 26 nt and can be designed on the basis of the information shown in SEQ ID NO: 1. Guidelines for designing effective siRNAs are known to the skilled person. Briefly, a short fragment of the target gene sequence (e.g., 19-40 nucleotides in length) is chosen as the target sequence of the siRNA of the invention. The short fragment of target gene sequence is a fragment of the target gene mRNA. In preferred embodiments, the criteria for choosing a sequence fragment from the target gene mRNA to be a candidate siRNA molecule include 1) a sequence from the target gene mRNA that is at least 50-100 nucleotides from the 5′ or 3′ end of the native mRNA molecule, 2) a sequence from the target gene mRNA that has a G/C content of between 30% and 70%, most preferably around 50%, 3) a sequence from the target gene mRNA that does not contain repetitive sequences (e.g., AAA, CCC, GGG, TTT, AAAA, CCCC, GGGG, TTTT), 4) a sequence from the target gene mRNA that is accessible in the mRNA, 5) a sequence from the target gene mRNA that is unique to the target gene, 6) avoids regions within 75 bases of a start codon. The sequence fragment from the target gene mRNA may meet one or more of the criteria identified above. The selected gene is introduced as a nucleotide sequence in a prediction program that takes into account all the variables described above for the design of optimal oligonucleotides. This program scans any mRNA nucleotide sequence for regions susceptible to be targeted by siRNAs. The output of this analysis is a score of possible siRNA oligonucleotides. The highest scores are used to design double stranded RNA oligonucleotides that are typically made by chemical synthesis. In addition to siRNA which is complementary to the mRNA target region, degenerate siRNA sequences may be used to target homologous regions. siRNAs according to the invention can be synthesized by any method known in the art. RNAs are preferably chemically synthesized using appropriately protected ribonucleoside phosphoramidites and a conventional DNA/RNA synthesizer. Additionally, siRNAs can be obtained from commercial RNA oligonucleotide synthesis suppliers.
siRNA molecules according to the aspects of the invention may be double stranded. In one embodiment, double stranded siRNA molecules comprise blunt ends. In another embodiment, double stranded siRNA molecules comprise overhanging nucleotides (e.g., 1-5 nucleotide overhangs, preferably 2 nucleotide overhangs). In some embodiments, the siRNA is a short hairpin RNA (shRNA); and the two strands of the siRNA molecule may be connected by a linker region (e.g., a nucleotide linker or a non-nucleotide linker). The siRNAs of the invention may contain one or more modified nucleotides and/or non-phosphodiester linkages. Chemical modifications well known in the art are capable of increasing stability, availability, and/or cell uptake of the siRNA. The skilled person will be aware of other types of chemical modification which may be incorporated into RNA molecules.
In one embodiment, recombinant DNA constructs as described in U.S. Pat. No. 6,635,805, incorporated herein by reference, may be used.
The silencing RNA molecule is introduced into the plant using conventional methods, for example a vector and Agrobacterium-mediated transformation. Stably transformed plants are generated and expression of the SOD7 and/or NGAL3 gene compared to a wild type control plant is analysed.
Silencing of the SOD7 and/or NGAL3 nucleic acid sequence may also be achieved using virus-induced gene silencing.
Thus, in one embodiment of the invention, the plant expresses a nucleic acid construct comprising a RNAi, shRNA snRNA, dsRNA, siRNA, miRNA, ta-siRNA, amiRNA or co-suppression molecule that targets the SOD7 or NGAL3 nucleic acid sequence as described herein and reduces expression of the endogenous SOD7 or NGAL3 nucleic acid sequence. A gene is targeted when, for example, the RNAi, snRNA, dsRNA, siRNA, shRNA miRNA, ta-siRNA, amiRNA or cosuppression molecule selectively decreases or inhibits the expression of the gene compared to a control plant. Alternatively, a RNAi, snRNA, dsRNA, siRNA, miRNA, ta-siRNA, amiRNA or cosuppression molecule targets A SOD7 or NGAL3 nucleic acid sequence when the RNAi, shRNA snRNA, dsRNA, siRNA, miRNA, ta-siRNA, amiRNA or cosuppression molecule hybridises under stringent conditions to the gene transcript.
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. 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.
In one embodiment, the suppressor nucleic acids may be anti-sense suppressors of expression of the NGAL2 or NGAL3 polypeptides. In using anti-sense sequences to down-regulate gene expression, a nucleotide sequence is placed under the control of a promoter in a “reverse orientation” such that transcription yields RNA which is complementary to normal mRNA transcribed from the “sense” strand of the target gene.
An anti-sense suppressor nucleic acid may comprise an anti-sense sequence of at least 10 nucleotides from the target nucleotide sequence. It may be preferable that there is complete sequence identity in the sequence used for down-regulation of expression of a target sequence, and the target sequence, although total complementarity or similarity of sequence is not essential. One or more nucleotides may differ in the sequence used from the target gene. Thus, a sequence employed in a down-regulation of gene expression in accordance with the present invention may be a wild-type sequence (e.g. gene) selected from those available, or a variant of such a sequence.
The sequence need not include an open reading frame or specify an RNA that would be translatable. It may be preferred for there to be sufficient homology for the respective anti-sense and sense RNA molecules to hybridise. There may be down regulation of gene expression even where there is about 5%, 10%, 15% or 20% or more mismatch between the sequence used and the target gene. Effectively, the homology should be sufficient for the down-regulation of gene expression to take place.
Suppressor nucleic acids may be operably linked to tissue-specific or inducible promoters. For example, integument and seed specific promoters can be used to specifically down-regulate a SOD7 or NGAL3 nucleic acids in developing ovules and seeds to increase final seed size.
Nucleic acid which suppresses expression of a NGAL2 or NGAL3 polypeptide as described herein may be operably linked to a heterologous regulatory sequence, such as a promoter, for example a constitutive, inducible, tissue-specific or developmental specific promoter. The construct or vector may be transformed into plant cells and expressed as described herein. Plant cells comprising such vectors are also within the scope of the invention.
In another aspect, the invention relates to a silencing construct to silence expression of NGAL2 or NGAL3 obtainable or obtained by a method as described herein and to a plant cell comprising such construct. Accordingly, the invention also relates to the use of a nucleic acid sequence comprising or consisting of SEQ ID NO: 1, 2 or 3 or a part thereof or a homologue of SEQ ID NO: 1, 2 or 3 or a part thereof in silencing expression of NGAL2 or NGAL3. Host cells transformed with such construct are also within the scope of the invention.
Recently, genome editing techniques have emerged as alternative methods to conventional mutagenesis methods (such as physical and chemical mutagenesis) or methods using the expression of transgenes in plants to produce mutant plants with improved phenotypes that are important in agriculture. These techniques employ sequence-specific nucleases (SSNs) including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the RNA-guided nuclease Cas9 (CRISPR/Cas9), which generate targeted DNA double-strand breaks (DSBs), which are then repaired mainly by either error-prone non-homologous end joining (NHEJ) or high-fidelity homologous recombination (HR). The SSNs have been used to create targeted knockout plants in various species ranging from the model plants, Arabidopsis and tobacco, to important crops, such as barley, soybean, rice and maize. Heritable gene modification has been demonstrated in Arabidopsis and rice using the CRISPR/Cas9 system and TALENs.
Targeted genome modification or targeted genome editing is a genome engineering technique that uses targeted DNA double-strand breaks (DSBs) to stimulate genome editing through homologous recombination (HR)-mediated recombination events. To achieve effective genome editing via introduction of site-specific DNA DSBs, four major classes of customizable DNA binding proteins can be used: meganucleases derived from microbial mobile genetic elements, ZF nucleases based on eukaryotic transcription factors, transcription activator-like effectors (TALEs) from Xanthomonas bacteria, and the RNA-guided DNA endonuclease Cas9 from the type II bacterial adaptive immune system CRISPR (clustered regularly interspaced short palindromic repeats). Meganuclease, ZF, and TALE proteins all recognize specific DNA sequences through protein-DNA interactions. Although meganucleases integrate its nuclease and DNA-binding domains, ZF and TALE proteins consist of individual modules targeting 3 or 1 nucleotides (nt) of DNA, respectively. ZFs and TALEs can be assembled in desired combinations and attached to the nuclease domain of Fokl to direct nucleolytic activity toward specific genomic loci.
Upon delivery into host cells via the bacterial type III secretion system, TAL effectors enter the nucleus, bind to effector-specific sequences in host gene promoters and activate transcription. Their targeting specificity is determined by a central domain of tandem, 33-35 amino acid repeats. This is followed by a single truncated repeat of 20 amino acids. The majority of naturally occurring TAL effectors examined have between 12 and 27 full repeats.
These repeats only differ from each other by two adjacent amino acids, their repeat-variable di-residue (RVD). The RVD that determines which single nucleotide the TAL effector will recognize: one RVD corresponds to one nucleotide, with the four most common RVDs each preferentially associating with one of the four bases. Naturally occurring recognition sites are uniformly preceded by a T that is required for TAL effector activity. TAL effectors can be fused to the catalytic domain of the Fokl nuclease to create a TAL effector nuclease (TALEN) which makes targeted DNA double-strand breaks (DSBs) in vivo for genome editing. The use of this technology in genome editing is well described in the art, for example in U.S. Pat. Nos. 8,440,431, 8,440,432 and 8,450,471. Reference 30 describes a set of customized plasmids that can be used with the Golden Gate cloning method to assemble multiple DNA fragments. As described therein, the Golden Gate method uses Type IIS restriction endonucleases, which cleave outside their recognition sites to create unique 4 bp overhangs. Cloning is expedited by digesting and ligating in the same reaction mixture because correct assembly eliminates the enzyme recognition site. Assembly of a custom TALEN or TAL effector construct and involves two steps: (i) assembly of repeat modules into intermediary arrays of 1-10 repeats and (ii) joining of the intermediary arrays into a backbone to make the final construct.
Another genome editing method that can be used according to the various aspects of the invention is CRISPR. The use of this technology in genome editing is well described in the art, for example in U.S. Pat. No. 8,697,359 and references cited herein. In short, CRISPR is a microbial nuclease system involved in defense against invading phages and plasmids. CRISPR loci in microbial hosts contain a combination of CRISPR-associated (Cas) genes as well as non-coding RNA elements capable of programming the specificity of the CRISPR-mediated nucleic acid cleavage (sgRNA). Three types (I-III) of CRISPR systems have been identified across a wide range of bacterial hosts. One key feature of each CRISPR locus is the presence of an array of repetitive sequences (direct repeats) interspaced by short stretches of non-repetitive sequences (spacers). The non-coding CRISPR array is transcribed and cleaved within direct repeats into short crRNAs containing individual spacer sequences, which direct Cas nucleases to the target site (protospacer). The Type II CRISPR is one of the most well characterized systems and carries out targeted DNA double-strand break in four sequential steps. First, two non-coding RNA, the pre-crRNA array and tracrRNA, are transcribed from the CRISPR locus. Second, tracrRNA hybridizes to the repeat regions of the pre-crRNA and mediates the processing of pre-crRNA into mature crRNAs containing individual spacer sequences. Third, the mature crRNA:tracrRNA complex directs Cas9 to the target DNA via Watson-Crick base-pairing between the spacer on the crRNA and the protospacer on the target DNA next to the protospacer adjacent motif (PAM), an additional requirement for target recognition. Finally, Cas9 mediates cleavage of target DNA to create a double-stranded break within the protospacer.
Cas9 is thus the hallmark protein of the type II CRISPR-Cas system, and a large monomeric DNA nuclease guided to a DNA target sequence adjacent to the PAM (protospacer adjacent motif) sequence motif by a complex of two noncoding RNAs: CRIPSR RNA (crRNA) and trans-activating crRNA (tracrRNA). The Cas9 protein contains two nuclease domains homologous to RuvC and HNH nucleases. The HNH nuclease domain cleaves the complementary DNA strand whereas the RuvC-like domain cleaves the non-complementary strand and, as a result, a blunt cut is introduced in the target DNA. Heterologous expression of Cas9 together with an sgRNA can introduce site-specific double strand breaks (DSBs) into genomic DNA of live cells from various organisms. For applications in eukaryotic organisms, codon optimized versions of Cas9, which is originally from the bacterium Streptococcus pyogenes, have been used.
The single guide RNA (sgRNA) is the second component of the CRISPR/Cas system that forms a complex with the Cas9 nuclease. sgRNA is a synthetic RNA chimera created by fusing crRNA with tracrRNA. The sgRNA guide sequence located at its 5′ end confers DNA target specificity. Therefore, by modifying the guide sequence, it is possible to create sgRNAs with different target specificities. The canonical length of the guide sequence is 20 bp. In plants, sgRNAs have been expressed using plant RNA polymerase III promoters, such as U6 and U3.
Using these techniques, it is possible to specifically target conserved domains to abolish the function of the NGAL2 and/or NGAL3 polypeptide.
For example, the conserved B3 domain or repression motif may be targeted.
Thus, in another embodiment of the invention directed to a mutant plant, plant cell, plant or a part thereof characterised in that the activity of a NGAL2 polypeptide is altered and said plant expresses a nucleic acid comprising a mutant SEQ ID NO. 1 or 2 and encoding a mutant NGAL2 polypeptide, a functional homologue or variant thereof, for example one which carries a mutation in the B3 or repressor domain.
Thus, in another embodiment of the invention directed to a mutant plant, plant cell, plant or a part thereof characterised in that the activity of a NGAL3 polypeptide is altered and said plant expresses a nucleic acid comprising a mutant SEQ ID NO. 4 and encoding a mutant NGAL3 polypeptide, a functional homologue or variant thereof which carries a mutation in the B3 or repressor domain.
In a preferred embodiment, the invention directed to a mutant plant, plant cell, plant or a part thereof characterised in that the activity of a NGAL2 and a NGAL3 polypeptide is altered and said plant expresses a nucleic acid comprising a mutant SEQ ID NO. 1 or 2 and encoding a mutant NGAL2 polypeptide, a functional homologue or variant thereof, for example one which carries a mutation in the B3 or repressor domain and said plant expresses a nucleic acid comprising a mutant SEQ ID NO. 4 and encoding a mutant NGAL3 polypeptide which carries a mutation in the B3 or repressor domain.
Mutations in the promoter region of SOD7 and/or NGAL3 resulting in a loss of function are also within the scope of the invention.
Constructs designed using the genome editing technologies to knock out or knock down NGAL2 or NGAL3, for example as shown herein, are also within the scope of the invention as well as host cells comprising these constructs. In one embodiment, the constructs comprise or consist of a sequence selected from SEQ ID NO: 155, 156, 157 or 158. Accordingly, in a further aspect of the invention, there is provided a nucleic acid construct comprising a sequence selected from SEQ ID NO: 155, 156, 157 or 158. In a further aspect of the invention, there is provided a nucleic acid construct comprising at least one CRISPR target sequence, wherein the target sequence is selected from SEQ ID Nos 150, 160, 161, 162 and 163. Preferably, the target sequence comprises at least two CRISPR target sequences, preferably SEQ ID No 159 and 160 or SEQ ID No 161 and 162, or SEQ ID No 161 and 163 or SEQ ID No 159 and 163.
In another embodiment of the methods of the invention, inactivating, repressing or down-regulating the activity of NGAL2 and/or NGAL3 can be achieved by manipulating the expression of SOD7 and/or NGAL3 inhibitors in a plant, for example transgenic plant. For example, a gene expressing a protein that inhibits the expression of the SOD7 and/or NGAL3 gene or activity of the SOD7 and/or NGAL3 protein can be introduced into a plant and over-expressed. The inhibitor may interact with the regulatory sequences that direct SOD7 and/or NGAL3 gene expression to down-regulate or repress SOD7 and/or NGAL3 gene expression. For example, the inhibitor may be a transcriptional repressor. Alternatively, it may interact and repress transcriptional regulators, for example transcription factors, that positively regulate expression of the SOD7 and/or NGAL3 gene. Alternatively, the inhibitor it may directly interact with the NGAL2 and/or NGAL3 protein to inhibit its activity or interact with modulators of the NGAL2 and/or NGAL3 protein. For example, the activity of the NGAL2 and/or NGAL3 protein may be inactivated, repressed or down-regulated by manipulating post-transcriptional modifications, of the NGAL2 and/or NGAL3 protein resulting in a reduced or lost activity.
In one embodiment, the methods of the invention comprise comparing the activity of the NGAL2 and/or NGAL3 polypeptide and/or expression of the SOD7 and/or NGAL3 gene with the activity of the NGAL2 and/or NGAL3 polypeptide and/or expression of the SOD7 and/or NGAL3 gene in a control plant.
In another aspect, the invention relates to a plant obtainable or obtained by a method as described herein.
In another aspect, the invention relates to an expression cassette comprising an isolated nucleic acid sequence comprising or consisting of a sequence as shown in SEQ ID NO: 1 or 2 a functional part, variant, homologue or orthologue thereof operably linked to a regulatory element. In another aspect, the invention relates to an expression cassette comprising an isolated nucleic acid sequence comprising or consisting of a sequence as shown in SEQ ID NO: 4 or a functional part, variant, homologue or orthologue thereof operably linked to a regulatory element. The regulatory element may be a promoter. The invention also relates to a vector comprising such expression cassette. The invention also relates to a composition comprising the two expression cassettes above.
In the methods described here, plants can be regenerated from plants transformed or genetically altered as described above and the phenotype, specifically the seed phenotype is analysed by known methods.
Transformation methods are known in the art. The nucleic acid sequence is introduced into said plant through a process called transformation. The 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 plants is now a routine technique in many species. 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, electroporation of protoplasts, microinjection into plant material, DNA or RNA-coated particle bombardment, infection with (non-integrative) viruses and the like. Transgenic plants, including transgenic crop plants, are preferably produced via Agrobacterium tumefaciens mediated transformation.
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 is 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 various aspects of the invention described herein clearly extend to any plant cell or any plant produced, obtained or obtainable by any of the methods described herein, and to all plant parts and propagules thereof unless otherwise specified. 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 extends to harvestable parts of a plant of the invention as described above such as, but not limited to seeds, leaves, fruits, flowers, stems, roots, rhizomes, tubers and bulbs. 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. The invention also relates to food products and food supplements comprising the plant of the invention or parts thereof.
While the foregoing disclosure provides a general description of the subject matter encompassed within the scope of the present invention, including methods, as well as the best mode thereof, of making and using this invention, the following examples are provided to further enable those skilled in the art to practice this invention and to provide a complete written description thereof. However, those skilled in the art will appreciate that the specifics of these examples should not be read as limiting on the invention, the scope of which should be apprehended from the claims and equivalents thereof appended to this disclosure. Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.
All documents mentioned in this specification are incorporated herein by reference in their entirety, including references to gene and protein accession numbers.
“and/or” where used herein is to be taken as specific disclosure of each of the multiple specified features or components with or without the other at each combination unless otherwise dictated. For example “A, B and/or C” is to be taken as specific disclosure of each of (i) A, (ii) B, (iii) C, (iv) A and B, (v) B and C or (vi) A and B and C, just as if each is set out individually herein.
Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.
The invention is further described in the following non-limiting examples.
EXAMPLESMethods
Plant Materials and Growth Conditions
Arabidopsis thaliana Columbia (Col-0) was used as wild-type line. The da1-1, sod7-1D, sod7-ko1 and ngal3-ko1 were in the Col-0 background. sod7-1D was identified as a suppressor of da1-1 by using T-DNA activation tagging method. The sod7-ko1 (SM_3_34191) and ngal3-ko1 (SM_3_36641) were identified in AtIDB (atidb.org) and obtained from Arabidopsis Stock Centre NASC collection. T-DNA insertions were confirmed by PCR and sequencing by using the primers described in Table 1. Arabidopsis plants were grown under long-day conditions (16 h light/8 h dark) at 22° C. Activation tagging screening The activation tagging plasmid pJFAT260 was introduced into the da1-1 mutant plants using Agrobacterium tumefaciens strain GV3101 (Fan et al., 2009; Fang et al., 2012), and T1 plants were selected by using the herbicide Basta. Seeds produced from T1 plants were used to isolate modifiers of da1-1.
Morphological and Cellular Analysis
To measure seed size, we photographed dry seeds of the wild type and mutants under a Leica microscope (LEICA S8APO) using Leica CCD (DFC420). The projective area of wild-type and mutant seeds was measured by using Image J software. Average seed weight was determined by weighing mature dry seeds in batches of 100 using an electronic analytical balance (METTLER TOLEDO AL104, China). The weights of five sample batches were measured for each seed lot. Fully expanded cotyledons, petals (stage 14) and leaves were scanned to produce digital images for area measurement. To measure cell number and cell size, petals, leaves, ovules and seeds were placed in a drop of clearing solution [30 ml H2O, 80 g Chloral hydrate (Sigma, C8383), 10 ml 100% Glycerol (Sigma, G6279)]. Cleared Samples were imaged under a Leica microscope (LEICA DM2500) with differential interference contrast (DIC) optics and photographed with a SPOT FLEX Cooled CCD Digital Imaging System. Area measurement was made by using Image J software.
Cloning of the SOD7 Gene
The flanking sequences of the T-DNA insertion of the sod7-1D mutant were identified by the thermal asymmetric interlaced PCR (TAIL-PCR) according to a previously reported method (Liu et al., 1995). Briefly, TAIL-PCR utilizes three nested specific primers (OJF22, OJF23 and OJF24) within the T-DNA region of the pJFAT260 vector and a shorter arbitrary degenerate primer (AD1). Thus, the relative amplification efficiencies of specific and non-specific products can be thermally controlled. TAIL-PCR products were sequenced using the primer OJF24. The specific primers OJF22, OJF23 and OJF24 and an arbitrary degenerate (AD1) primer are described in Table 1.
Constructs and Plant Transformation
The 35S:GFP-SOD7, pSOD7:SOD7-GFP and pSOD7:GUS constructs were made using a PCR-based Gateway system. The coding sequence (CDS) of SOD7 was amplified using the primers SOD7CDS-F and SOD7CDS-R (Table 1). PCR products were cloned into pCR8/TOPO TA cloning vector. The SOD7 CDS was then subcloned into the binary vector pMDC43 with the GFP gene to generate the transformation plasmid 35S:GFP-SOD7. The SOD7 genomic sequence containing 2040-bp promoter sequence and 2104-bp SOD7 gene was amplified using the primers SOD7G-F and SOD7G-R (Table 1). PCR products were cloned into pCR8/TOPO TA cloning vector. The SOD7 genomic sequence was then subcloned into the binary vectors pMDC107 with the GFP gene to generate the transformation plasmid pSOD7:SOD7-GFP. The 2262-bp SOD7 promoter sequence was amplified using the primers SOD7P-F and SOD7P-R (Table 1). PCR products were cloned into pCR8/TOPO TA cloning vector. The SOD7 promoter was then subcloned into the binary vectors pGWB3 with the GUS gene to generate the transformation plasmid pSOD7:GUS. The plasmids 35S:GFP-SOD7, pSOD7:SOD7-GFP and pSOD7:GUS were introduced into Col-0 or sod7-ko1 ngal3ko1 plants using Agrobacterium tumefaciens GV3101, respectively, and transformants were selected on hygromycin (30 μg/ml)-containing medium. The SOD7 cDNA was cloned into the Apal and Spel sites of the binary vector pER8 to generate a chemically inducible construct pER8-SOD7. The specific primers for the pER8-SOD7 construct were SOP7ER-F and SOD7ER-R. The plasmid pER8-SOD7 was introduced into Col-0 plants using Agrobacterium tumefaciens GV3101, and transformants were selected on hygromycin (30 μg/ml)-containing medium. GUS staining Samples (pSOD7:GUS) were stained in a GUS staining solution (1 mM X-gluc, 50 Mm NaPO4 buffer, 0.4 mM each K3Fe(CN)6/K4Fe(CN)6, and 0.1% (v/v) Triton X-100) and incubated at 37° C. for 3 hours. After GUS staining, chlorophyll was removed by 70% ethanol. RT-PCR and quantitative real-time RT-PCR. Total RNA was extracted from Arabidopsis seedlings using an RNAprep pure Plant kit (TIANGEN). mRNA was reverse transcribed into cDNA using SuperScriptIII reverse transcriptase (Invitrogen). cDNA samples were standardized on ACTIN2 transcript amount using the primers ACTIN2-F and ACTIN2-R (Table 1). Quantitative real-time RT-PCR analysis was performed with a Lightcycler 480 machine (Roche) using the Lightcycler 480 SYBR Green I Master (Roche). ACTIN2 mRNA was used as an internal control, and relative amounts of mRNA were calculated using the comparative threshold cycle method. The primers used for RT-PCR and quantitative real-time RT-PCR are described in Table 1.
The Chromatin Immunoprecipitation (ChIP) Assay
The chromatin immunoprecipitation (ChIP) assay was performed as described previously with minor modifications (Gendrel et al., 2005). Briefly, 35S:GFP and 35S:GFP-SOD7 transgenic seeds were grown on ½ MS plates for 10 days. The seedlings were cross-linked by 1% formaldehyde for 15 min in vacuum and stopped by 0.125 M Glycine. Samples were ground in liquid nitrogen, and nuclei were isolated. Chromatin was immunoprecipitated by anti-GFP (Roche, 11814460001) and protein A+G beads (Millpore Magna ChIP Protein A+G Magnetic Beads, 16-663). DNA was precipitated by glycogen, NaOAc and ethanol, washed by 70% ethanol, and dissolved in 60 μl of water. Gene-specific primers (PF1-F, PF1-R, PF-2F, PF2-R, ACTIN7-ChIP-F, and ACTIN7-ChIP-R) were used to quantify the enrichment of each fragment (Table 1).
The DNA Electrophoretic Mobility Shift Assay (EMSA)
The coding sequence of SOD7 was cloned into the NdeI and BamHI sites of the pMAL-C2 vector to generate the construct MBP-SOD7. MBP-SOD7 fusion proteins were expressed in Escherichia coli BL21 (DE3) (Biomed) and purified by Amylose resins(New England Biolabs). The biotin-labeled and unlabeled probes were synthesized as forward and reverse strands. The forward and reverse strands were then incubated in a solution (50 mM Tris-HCl, 5 mM EDTA and 250 mM NaCl) at 95° C. for 10 min and renatured to double stranded probes at room temperature. The gel-shift assay was performed according to the method described previously (Smaczniak et al., 2012).
Results
sod7-1D Suppresses the Seed Size Phenotype of Dal-1
We previously identified the ubiquitin receptor DA1 as a negative regulator of seed size in Arabidopsis (Li et al., 2008). The da1-1 mutant formed large seeds due to increased cell proliferation in the maternal integuments (Li et al., 2008; Xia et al., 2013). To identify novel components in the DA1 pathway or other seed size regulators, we initiated a T-DNA activation tagging screen for modifiers of da1-1 (Fang et al., 2012). A dominant suppressor of da1-1 (sod7-1D) was isolated from seeds produced from approximate 16,000 T1 plants (
sod7-1D Produces Small Seeds
We isolated the single sod7-1D mutant among F2 progeny derived from a cross between the wild type (Col-0) and sod7-1D da1-1. The sod7-1D seeds were significantly smaller and lighter than wild-type seeds (
SOD7 Encodes a B3 Domain Transcriptional Repressor NGAL2
To determine whether the seed and organ size phenotypes of sod7-1D was caused by the T-DNA insertion, we firstly analyzed the genetic linkage of the mutant phenotypes with Basta resistance, which is conferred by the selectable marker of the activation tagging vector (Fan et al., 2009). In a T2 population, 181 plants with sod7-1D da1-1 phenotypes were resistant, whereas 55 plants with da1-1 phenotypes were sensitive, indicating that the insertion is cosegregated with the sod7-1D phenotypes. To clone the SOD7 gene, we isolated the T-DNA flanking sequences using thermal asymmetric interlaced PCR (Liu et al., 1995). DNA sequencing revealed that the T-DNA had inserted approximately 5.6 kb upstream of the At3g11580 and about 3.7 kb upstream of the At3g11590 gene (
Expression Pattern and Subcellular Localization of SOD7
To monitor SOD7 expression pattern during development, the pSOD7:GUS and pSOD7:SOD7-GFP vectors were constructed and transformed to wild-type plants, respectively. The tissue-specific expression patterns of SOD7 were examined using a histochemical assay for GUS activity. In seedlings, relatively higher GUS activity was detected in younger leaves than in older leaves (
SOD7/NGAL2 Acts Redundantly with NGAL3 to Control Seed Size
In order to further investigate the function of SOD7 in seed size control, we isolated T-DNA inserted loss-of-function mutants for SOD7 and NGAL3, the most closely related family member. sod7-ko1 (SM_3_34191) was identified with T-DNA insertion in the first exon of the SOD7 gene (
SOD7 Acts Maternally to Control Seed Size
As the size of a seed is determined by the zygotic and/or maternal tissues (Garcia et al., 2005; Xia et al., 2013; Du et al., 2014), we asked whether SOD7 functions maternally or zygotically. We therefore performed reciprocal cross experiments between the wild type and sod7-ko1 ngal3-ko1. The effect of sod7-ko1 ngal3-ko1 on seed size was observed only when sod7-ko1 ngal3-ko1 was used as maternal plants (
SOD7 Regulates Cell Proliferation in the Maternal Integuments
The reciprocal crosses showed that SOD7 functions maternally to influence seed size. The integuments surrounding the ovule are maternal tissues, which could set the growth potential of the seed coat after fertilization. Consistent with this idea, several studies showed that the integument size influences the final size of seeds in Arabidopsis (Garcia et al., 2005; Schruff et al., 2006; Adamski et al., 2009; Xia et al., 2013; Du et al., 2014). We therefore asked whether SOD7 acts through the maternal integuments to determine seed size. To test this, we characterized mature ovules of the wild type and sod7-ko1 ngal3-ko1. As shown in
SOD7 Acts in a Common Pathway with KLU to Control Seed Size, but does so Independently of DA1
The Arabidopsis klu mutants formed small seeds due to the decreased cell proliferation in the integuments, while plants overexpressing KLU/CYP78A5 produced large seeds as a result of the increased cell proliferation in the integuments (Adamski et al., 2009), suggesting that SOD7 and KLU could function antagonistically in a common pathway to control seed growth. To test for genetic interactions between SOD7 and KLU, we generated the klu-4 sod7-ko1 ngal3-ko1 triple mutant and measured the size of seeds from wild-type, klu-4, sod7-ko1 ngal3-ko1 and klu-4 sod7-ko1 ngal3-ko1 plants. As shown in
Thus, these genetic analyses show that klu-4 is epistatic to sod7-ko1 ngal3-ko1 with respect to seed and organ size, indicating that SOD7 and KLU act antagonistically in a common pathway to control seed and organ growth. To further understand the cellular basis of epistatic interactions between SOD7 and KLU, we investigated the outer integument cell number of ovules and developing seeds from wild-type, klu-4, sod7-ko1 ngal3-ko1 and klu-4 sod7-ko1 ngal3-ko1 plants. The number of outer integument cells in klu-4 sod7-ko1 ngal3-ko1 ovules was similar to that in klu-4 ovules (
Considering that sod7-1D was identified as a suppressor of da1-1 in seed size, we further asked whether SOD7 and DA1 could act in the same genetic pathway. To test this, we measured the size of wild-type, da1-1, sod7-1D and sod7-1D da1-1 seeds. The genetic interaction between sod7-1D and da1-1 was essentially additive for seed size, compared with that of sod7-1D and da1-1 single mutants, indicating that SOD7 might function independently of DA1 to control seed size. We further crossed sod7-ko1 ngal3-ko1 with da1-1 and generated the sod7-ko1 ngal3-ko1 da1-1 triple mutant and measured its seed size. The genetic interaction between sod7-ko1 ngal3-ko1 and da1-1 was also additive for seed size, compared with their parental lines, further supporting that SOD7 functions to control seed growth separately from DA1.
SOD7 Directly Binds to the Promoter of KLU and Represses the Expression of KLU
Considering that SOD7 acts antagonistically in a common pathway with KLU to control seed size, we asked whether the transcription repressor SOD7 could repress the expression of KLU. We therefore investigated the expression of KLU in the chemically-inducible SOD7 (pER8-SOD7) transgenic plants. After the pER8-SOD7 transgenic plants were treated with the inducer (β-estradiol), the expression of SOD7 was strongly induced at 4 and 8 hours (
To determine whether SOD7 can directly bind to the promoter of the KLU gene, we performed a chromatin immunoprecipitation (ChIP) assay with 35S:GFP and 35:GFP-SOD7 transgenic plants. It has been reported that the CACCTG sequence is recognized by the B3 domain of RAV1, one member of the RAV family (Kagaya et al., 1999; Yamasaki et al., 2004). We therefore analyzed the promoter sequence of KLU and did not find an intact CACCTG sequence within 2 kb promoter region of KLU. However, we found a similar sequence (CACTTG) in the promoter region of KLU (
Discussion
Seed size is crucial for plant fitness and agricultural purposes, but little is known about the genetic and molecular mechanisms that set the final size of seeds in plants. In this study, we show that SOD7 acts maternally to control seed size by restricting cell proliferation in the integuments of ovules and developing seeds. SOD7 encodes a B3 domain transcriptional repressor NGAL2 and acts redundantly with its closest homolog NGAL3 to control seed size. Genetic analyses indicate that SOD7 functions in a common pathway with the maternal factor KLU to control seed growth, but does so independently of DA1. Further results reveal that SOD7 directly binds to the promoter region of KLU and represses KLU expression. Thus, our findings identify SOD7 as a negative factor for seed size and define the genetic and molecular mechanisms of SOD7 and KLU in seed size control.
SOD7 Acts Maternally to Regulate Seed Size
The sod7-1D gain-of-function mutant was identified as a suppressor of the large seed phenotype of da1-1. However, genetic analyses showed that SOD7 functions independently of DA1 to control seed growth. The sod7-1D single mutant produced small seeds and organs (
Reciprocal cross experiments showed that SOD7 acts maternally to restrict seed growth, and the endosperm and embryo genotypes for SOD7 do not determine seed size (
small seeds in Arabidopsis (Adamski et al., 2009; Du et al., 2014). Considering that cells in the integuments mainly undergo expansion after fertilization (Garcia et al., 2005), it is possible that the number of cells in the integuments determines the growth potential of the seed coat after fertilization.
The Genetic and Molecular Mechanisms of SOD7 and KLU in Seed Size Control
The sod7-1D mutant had small seeds and organs (
SOD7 encodes a B3 domain transcriptional repressor NGAL2 that is localized in nuclei of Arabidopsis cells (
For many plants, the seeds are the main product to be harvested, and an increase in seed size would be beneficial for growers. In this study, we identify SOD7 as a negative regulator of seed size, and demonstrate that SOD7 acts in a common genetic pathway with KLU to control seed size. Our current knowledge of SOD7 functions suggests that the SOD7 gene (and its homologs in other plant species) could be used to engineer large seed size in crops. Considering that crop plants have undergone selection for large seed size during domestication (Fan et al., 2006; Song et al., 2007; Gegas et al., 2010), it will be a worthwhile challenge to know whether beneficial alleles of the SOD7 gene have already been utilized by plant breeders.
Knockout Experiments in Rice Using Genome Editing
Genome editing experiments to knock out os11g01560000 and/or Os12g0157000 in rice are being carried out using the crisper-cas9 system. Four vectors, each with two recognition (CRISPR target) sites, have been constructed, to achieve these knock outs, as described in
1. The target sites were identified. The target site should be (or approximately so) 20 nucleotides before a NGG sequence, N being for any nucleotide. The target sequence was then evaluated using the website: http://cbi.hzau.edu.cn/crispr/help.php (incorporated herein by reference). Of note, the target site should be unique in the genome.
2. Using overlap PCR, the target sequence is linked with the U6 sequence, as shown in
3. Using infusion technology we connected the U6-guide-gRNA scaffold fragment to the vector pMDC99-cas9 to obtain the pMDC99-cas9-U6-guide-gRNA scaffold constructs. These constructs were named zyy1,zyy2, zyy3,zyy4. The full sequences of these constructs are represented in SEQ ID NO: 155, 156, 157 and 158 respectively. Each construct contains two recognition sites, which are highlighted in the sequence information, and are represented separately as SEQ ID Nos 159, 160, 161, 162 and 163.
4. We then transformed these constructs into Agrobacteria and used an Agrobacteria mediated method to transform rice and obtain gene-edited rice. Transformation of plants is a routine technique that is well known to the skilled person. Nonetheless, a brief outline of transformation techniques is provided above.
Knock out lines are being analysed to assess the phenotype.
- Adamski, N. M., Anastasiou, E., Eriksson, S., O'Neill, C. M., and Lenhard, M. (2009). mLocal maternal control of seed size by KLUH/CYP78A5-dependent growth signaling. Proceedings of the National Academy of Sciences of the United States of America 106, 20115-20120.
- Alvarez, J. P., Goldshmidt, A., Efroni, I., Bowman, J. L., and Eshed, Y. (2009). Th NGATHA distal organ development genes are essential for style specification in Arabidopsis. Plant Cell 21, 1373-1393.
- Anastasiou, E., Kenz, S., Gerstung, M., MacLean, D., Timmer, J., Fleck, C., and Lenhard, M. (2007). Control of plant organ size by KLUH/CYP78A5-dependent intercellular signaling. Developmental cell 13, 843-856.
- Cheng, Z. J., Zhao, X. Y., Shao, X. X., Wang, F., Zhou, C., Liu, Y. G., Zhang, Y., and Zhang, X. S. (2014). Abscisic Acid Regulates Early Seed Development in Arabidopsis by AB15-Mediated Transcription of SHORT HYPOCOTYL UNDER BLUE1. Plant Cell 26, 1053-1068.
- Du, L., Li, N., Chen, L., Xu, Y., Li, Y., Zhang, Y., and Li, C. (2014). The Ubiquitin Receptor DA1 Regulates Seed and Organ Size by Modulating the Stability of the Ubiquitin-Specific Protease UBP15/SOD2 in Arabidopsis. Plant Cell 26, 665-677.
- Engelhorn, J., Reimer, J. J., Leuz, I., Gobel, U., Huettel, B., Farrona, S., and Turck, F. (2012). Development-related PcG target in the apex 4 controls leaf margin architecture in Arabidopsis thaliana. Development 139, 2566-2575.
- Fan, C., Xing, Y., Mao, H., Lu, T., Han, B., Xu, C., Li, X., and Zhang, Q. (2006). GS3, a major QTL for grain length and weight and minor QTL for grain width and thickness in rice, encodes a putative transmembrane protein. Theor Appl Genet 112, 1164-1171.
- Fan, J., Hill, L., Crooks, C., Doerner, P., and Lamb, C. (2009). Abscisic acid has a key role in modulating diverse plant-pathogen interactions. Plant physiology 150, 1750-1761.
- Fang, W., Wang, Z., Cui, R., Li, J., and Li, Y. (2012). Maternal control of seed size by EOD3/CYP78A6 in Arabidopsis thaliana. Plant J 70, 929-939.
- Garcia, D., Fitz Gerald, J. N., and Berger, F. (2005). Maternal control of integument cell elongation and zygotic control of endosperm growth are coordinated to determine seed size in Arabidopsis. Plant Cell 17, 52-60.
- Garcia, D., Saingery, V., Chambrier, P., Mayer, U., Jurgens, G., and Berger, F. (2003). Arabidopsis haiku mutants reveal new controls of seed size by endosperm. Plant physiology 131, 1661-1670.
- Gegas, V. C., Nazari, A., Griffiths, S., Simmonds, J., Fish, L., Orford, S., Sayers, L., Doonan, J. H., and Snape, J. W. (2010). A genetic framework for grain size and shape variation in wheat. Plant Cell 22, 1046-1056.
- Gendrel, A. V., Lippman, Z., Martienssen, R., and Colot, V. (2005). Profiling histone modification patterns in plants using genomic tiling microarrays. Nat Methods 2, 213-218.
- Harper, J. L., Lovell, P. H., and Moore, K. G. (1970). The Shapes and Sizes of Seeds. Annual Review of Ecology and Systematics 1, 327-356
- Ikeda, M., and Ohme-Takagi, M. (2009). A novel group of transcriptional repressors in Arabidopsis. Plant & cell physiology 50, 970-975.
- Jofuku, K. D., Omidyar, P. K., Gee, Z., and Okamuro, J. K. (2005). Control of seed mass and seed yield by the floral homeotic gene APETALA2. Proceedings of the National Academy of Sciences of the United States of America 102, 3117-3122.
- Kagaya, Y., Ohmiya, K., and Hattori, T. (1999). RAV1, a novel DNA-binding protein, binds to bipartite recognition sequence through two distinct DNA-binding domains uniquely found in higher plants. Nucleic Acids Res 27, 470-478.
- Kang, X., Li, W., Zhou, Y., and Ni, M. (2013). A WRKY transcription factor recruits the SYG1-like protein SHB1 to activate gene expression and seed cavity enlargement. PLoS Genet 9, e1003347.
- Li, J., Nie, X., Tan, J. L., and Berger, F. (2013). Integration of epigenetic and genetic controls of seed size by cytokinin in Arabidopsis. Proceedings of the National Academy of Sciences of the United States of America 110, 15479-15484.
- Li, Y., Zheng, L., Corke, F., Smith, C., and Bevan, M. W. (2008). Control of final seed and organ size by the DA1 gene family in Arabidopsis thaliana. Genes Dev 22, 1331-1336.
- Liu, Y. G., Mitsukawa, N., Oosumi, T., and Whittier, R. F. (1995). Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR. Plant J 8, 457-463.
- Lopes, M. A., and Larkins, B. A. (1993). Endosperm origin, development, and function. Plant Cell 5, 1383-1399.
- Luo, M., Dennis, E. S., Berger, F., Peacock, W. J., and Chaudhury, A. (2005). MINISEED3 (MINI3), a WRKY family gene, and HAIKU2 (IKU2), a leucine-rich repeat (LRR) KINASE gene, are regulators of seed size in Arabidopsis. Proceedings of the National Academy of Sciences of the United States of America 102, 17531-17536.
- Moles, A. T., Ackerly, D. D., Webb, C. O., Tweddle, J. C., Dickie, J. B., and Westoby, M. (2005). A brief history of seed size. Science 307, 576-580.
- Ohto, M. A., Fischer, R. L., Goldberg, R. B., Nakamura, K., and Harada, J. J. (2005). Control of seed mass by APETALA2. Proceedings of the National Academy of Sciences of the United States of America 102, 3123-3128.
- Ohto, M. A., Floyd, S. K., Fischer, R. L., Goldberg, R. B., and Harada, J. J. (2009). Effects of APETALA2 on embryo, endosperm, and seed coat development determine seed size in Arabidopsis. Sex Plant Reprod 22, 277-289.
- Orsi, C. H., and Tanksley, S. D. (2009). Natural variation in an ABC transporter gene associated with seed size evolution in tomato species. PLoS Genet 5, e1000347.
- Schruff, M. C., Spielman, M., Tiwari, S., Adams, S., Fenby, N., and Scott, R. J. (2006). The AUXIN RESPONSE FACTOR 2 gene of Arabidopsis links auxin signalling, cell division, and the size of seeds and other organs. Development 133, 251-261. Scott, R. J., Spielman, M., Bailey, J., and Dickinson, H. G. (1998). Parent-of-origin effects on seed development in Arabidopsis thaliana. Development 125, 3329-3341.
- Smaczniak, C., Immink, R. G., Muino, J. M., Blanvillain, R., Busscher, M., Busscher-Lange, J., Dinh, Q. D., Liu, S., Westphal, A. H., Boeren, S., Percy, F., Xu, L., Caries, C. C., Angenent, G. C., and Kaufmann, K. (2012). Characterization of MADS-domain transcription factor complexes in Arabidopsis flower development. Proceedings of the National Academy of Sciences of the United States of America 109, 1560-1565.
- Song, X. J., Huang, W., Shi, M., Zhu, M. Z., and Lin, H. X. (2007). A QTL for rice grain width and weight encodes a previously unknown RING-type E3 ubiquitin ligase. Nat Genet 39, 623-630.
- Swaminathan, K., Peterson, K., and Jack, T. (2008). The plant B3 superfamily. Trends Plant Sci 13, 647-655.
- Trigueros, M., Navarrete-Gomez, M., Sato, S., Christensen, S. K., Pelaz, S., Weigel, D., Yanofsky, M. F., and Ferrandiz, C. (2009). The NGATHA genes direct style development in the Arabidopsis gynoecium. Plant Cell 21, 1394-1409.
- Wang, A., Garcia, D., Zhang, H., Feng, K., Chaudhury, A., Berger, F., Peacock, W. J., Dennis, E. S., and Luo, M. (2010). The VQ motif protein IKU1 regulates endosperm growth and seed size in Arabidopsis. Plant J 64, 670-679.
- Westoby, M., Falster, D. S., Moles, A. T., Vesk, P. A., and Wright, I. J. (2002). PLANT ECOLOGICAL STRATEGIES: Some Leading Dimensions of Variation Between Species. Annual Review of Ecology and Systematics 33, 125-159.
- Xia, T., Li, N., Dumenil, J., Li, J., Kamenski, A., Bevan, M. W., Gao, F., and Li, Y. (2013). The Ubiquitin Receptor DA1 Interacts with the E3 Ubiquitin Ligase DA2 to Regulate Seed and Organ Size in Arabidopsis. Plant Cell 25, 3347-3359.
- Xiao, W., Brown, R. C., Lemmon, B. E., Harada, J. J., Goldberg, R. B., and Fischer, R. L. (2006). Regulation of seed size by hypomethylation of maternal and paternal genomes. Plant physiology 142, 1160-1168.
- Xu, R., and Li, Y. (2011). Control of final organ size by Mediator complex subunit 25 in Arabidopsis thaliana. Development 138, 4545-4554.
- Yamasaki, K., Kigawa, T., Inoue, M., Tateno, M., Yamasaki, T., Yabuki, T., Aoki, M., Seki, E., Matsuda, T., Tomo, Y., Hayami, N., Terada, T., Shirouzu, M., Osanai, T., Tanaka, A., Seki, M., Shinozaki, K., and Yokoyama, S. (2004). Solution structure of the B3 DNA binding domain of the Arabidopsis cold-responsive transcription factor RAV1. Plant Cell 16, 3448-3459.
- Zhou, Y., Zhang, X., Kang, X., Zhao, X., and Ni, M. (2009). SHORT HYPOCOTYL UNDER BLUE1 associates with MINISEED3 and HAIKU2 promoters in vivo to regulate Arabidopsis seed development. Plant Cell 21, 106-117.
Claims
1.-16. (canceled)
17. A method for altering a plant phenotype comprising reducing or abolishing the expression of a nucleic acid sequence encoding a NGAL2 polypeptide or reducing or abolishing the activity of a NGAL2 polypeptide, or reducing or abolishing the expression of a nucleic acid sequences encoding a NGAL3 polypeptide, or reducing or abolishing the activity of a NGAL3 polypeptide, or reducing or abolishing the expression of nucleic acid sequences encoding NGAL2 and NGAL3 polypeptides or reducing or abolishing the activity of a NGAL2 and NGAL3 polypeptide, relative to a control plant.
18.-22. (canceled)
23. The method according to claim 17, wherein the NGAL2 polypeptide comprises SEQ D NO: 3, a functional variant or homologue thereof.
24. The method according to claim 17, wherein the nucleic acid sequence encoding a NGAL2 polypeptide comprises SEQ ID NO: 1 or 2, a functional variant or homologue thereof.
25. The method according to claim 24 wherein the functional variant or homologue comprises a nucleic acid sequence as shown in SEQ ID NO: 49-145.
26. The method according to claim 17, wherein the NGAL3 polypeptide comprises SEQ ID NO: 5, a functional variant or homologue thereof.
27. The method according to claim 17 wherein the NGAL3 nucleic acid sequence encoding a NGAL3 polypeptide comprises SEQ ID NO: 4, a functional variant or homologue thereof.
28. The method according to claim 27 wherein the functional variant or homologue comprises SEQ ID NOs:49-145.
29.-33. (canceled)
34. The method according to claim 17, wherein said phenotype is characterised by increased seed size relative to a control plant.
35.-36. (canceled)
37. A vector comprising SEQ ID NO: 1, 2 or 3 or a functional variant or homolog thereof.
38. (canceled)
Type: Application
Filed: Jul 6, 2020
Publication Date: Nov 12, 2020
Inventor: YUNHAI LI (BEIJING)
Application Number: 16/946,783
