POLYSACCHARIDE SYNTHASES (H)
The present invention relates generally to polysaccharide synthases. More particularly, the present invention relates to (1,3;1,4)-β-D-glucan synthases. The present invention provides, among other things, methods for influencing the level of (1,3;1,4)-β-D-glucan produced by a cell and nucleic acid and amino acid sequences which encode (1,3;1,4)-β-D-glucan synthases.
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The present invention claims priority to Australian provisional patent application 2007907071 the content of which is hereby incorporated by reference.
FIELD OF THE INVENTIONThe present invention relates generally to polysaccharide synthases. More particularly, the present invention relates to (1,3;1,4)-β-
The various tissues of cereal grains have diverse functions during grain development, dormancy and after germination.
For example, the pericarp and seed coat tissues are concerned with the protection of the seed during development and during dormancy. However, by grain maturity, these outer grain tissues have died and the tissue residues consist almost entirely of cell wall residues. The nucellar tissue between the seed coat and the aleurone surface is involved in transfer of nutrients to the developing grain, however, at maturity this tissue has also collapsed to leave cell wall remnants. The thin walled cells of the starchy endosperm of mature grain are dead, but are packed with starch and storage protein. In contrast, the thick-walled, nucleated, aleurone cells are alive at grain maturity, and are packed with protein bodies and lipid droplets. At the interface of the starchy endosperm lies the scutellum, which functions in delivering nutrients to the developing endosperm and, during germination, transfers digestion products of the endosperm reserves to the developing embryo.
The different structure and function of each tissue type in the grain is determined, at least in part, by the cell wall composition of each of these cell types.
Non-cellulosic polysaccharides are key components in the cell walls of cereal grain tissues and include, for example, (1,3;1,4)-β-
Although the precise physical relationships between individual non-cellulosic polysaccharides and other wall components have not been described, it is generally considered that in the wall, microfibrils of cellulose are embedded in a matrix phase of non-cellulosic polysaccharides and protein. Wall integrity is maintained predominantly through extensive non-covalent interactions, especially hydrogen bonding, between the matrix phase and microfibrillar constituents. In the walls of some grain tissues covalent associations between heteroxylans, lignin and proteins are present. The extent of covalent associations between components also varies with the wall type and genotype.
Non-cellulosic polysaccharides, especially heteroxylans and (1,3;1,4)-β-
In contrast, in the cell walls of the pericarp-seed coat, which provides a protective coat for the embryo and endosperm and which is not mobilized during germination, cellulose and lignin contents are much higher and the concentrations of non-cellulosic polysaccharides are correspondingly lower.
(1,3;1,4)-β-
These non-cellulosic polysaccharides are important constituents of the walls of the starchy endosperm and aleurone cells of most cereal grains, where they can account for up to 70%-90% by weight of the cell walls.
Barley, oat and rye grains are rich sources of (1,3;1,4)-β-
(1,3;1,4)-β-
However, despite this interest, major gaps remain in our knowledge of the genes and enzymes that control non-cellulosic polysaccharide biosynthesis, including (1,3;1,4)-β-
(1,3;1,4)-β-
Identification of the genes encoding (1,3;1,4)-β-
Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country.
SUMMARY OF THE INVENTIONIn accordance with the present invention, nucleotide sequences and corresponding amino acid sequences that encode a family of (1,3;1,4)-β-
As a result of the identification of nucleotide sequences and corresponding amino acid sequences that encode (1,3;1,4)-β-
Therefore, in a first aspect, the present invention provides a method for modulating the level of (1,3;1,4)-β-
In some embodiments, the level and/or activity of a (1,3;1,4)-β-
In some embodiments, the present invention contemplates increasing the level of (1,3;1,4)-β-
The present invention also facilitates the production of (1,3;1,4)-β-
In a fourth aspect, the present invention also provides (1,3;1,4)-β-
In a fifth aspect, the present invention also provides a cell comprising:
-
- a modulated level and/or activity of CslH-encoded (1,3;1,4)-β-
D -glucan synthase relative to a wild type cell of the same taxon; and/or - modulated expression of a CslH nucleic acid relative to a wild type cell of the same taxon.
- a modulated level and/or activity of CslH-encoded (1,3;1,4)-β-
In some embodiments, the cell further comprises a modulated level of (1,3;1,4)-β-
Furthermore, in a sixth aspect, the present invention provides a multicellular structure comprising one or more cells according to the fifth aspect of the invention.
The present invention also provides cereal grain comprising one or more cells according to the fifth aspect of the invention. Therefore, in a seventh aspect, the present invention provides a cereal grain comprising a modulated level of (1,3;1,4)-β-
In an eighth aspect, the present invention also provides flour comprising:
-
- flour produced by the milling of the grain of the seventh aspect of the invention; and
- optionally, flour produced by the milling of one or more other grains.
As set out above, the present invention is predicated, in part, on the identification and isolation of CslH nucleotide sequences and CslH amino acid sequences that encode (1,3;1,4)-β-
Therefore, in a ninth aspect, the present invention provides an isolated CslH nucleic acid or a complement, reverse complement or fragment thereof.
In a tenth aspect, the present invention provides a genetic construct or vector comprising an isolated nucleic acid molecule of the ninth aspect of the invention.
In an eleventh aspect, the present invention provides a cell comprising the isolated nucleic acid molecule of the ninth aspect of the invention or genetic construct of the tenth aspect of the invention.
In a twelfth aspect, the present invention provides a multicellular structure comprising one or more of the cells of the eleventh aspect of the invention.
As set out above, the present invention also provides amino acid sequences for CslH-encoded (1,3;1,4)-β-
Accordingly, in a thirteenth aspect, the present invention provides an isolated polypeptide comprising an amino acid sequence defining a CslH-encoded (1,3;1,4)-β-
In a fourteenth aspect, the present invention provides an antibody or an epitope binding fragment thereof, raised against an isolated CslH-encoded (1,3;1,4)-β-
Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.
Nucleotide and amino acid sequences are referred to herein by a sequence identifier number (SEQ ID NO:). The SEQ ID NOs: correspond numerically to the sequence identifiers <400>1 (SEQ ID NO: 1), <400>2 (SEQ ID NO: 2), etc. A summary of the sequence identifiers is provided in Table 1. A sequence listing is provided at the end of the specification.
It is to be understood that following description is for the purpose of describing particular embodiments only and is not intended to be limiting with respect to the above description.
The present invention is predicated, in part, on the identification of genes which encode biosynthetic enzymes for (1,3;1,4)-β-
“(1,3;1,4)-β-
The ratio of (1→4)- to (1→3)-linkages in naturally occurring (1,3;1,4)-β-
The ratio of tri- to tetra-saccharide units in endogenous (1,3;1,4)-β-
The average molecular masses reported for cereal (1,3;1,4)-β-
The extractability of (1,3;1,4)-β-
For example, a portion of the (1,3;1,4)-β-
As mentioned above, the present invention is predicated, in part, on the identification of biosynthetic enzymes, and their encoding genes, that catalyse the synthesis of (1,3;1,4)-β-
The present invention arises, in part, from an analysis of expressed sequence tag libraries and other sequence databases including cellulose synthase (CesA) genes. More particularly, it was noted in these analyses that the CesA genes were in fact members of a much larger super-family of genes, which included both the CesA genes and the cellulose synthase-like (Csl) gene family.
The Csl gene families in most vascular plants are very large and have been divided into several groups, designated CslA to CslH. In Arabidopsis thaliana there are 29 known Csl genes and in rice about 37. Overall, the Arabidopsis genome is believed to contain more than 700 genes involved in cell wall metabolism. However, in general, the specific functions of these genes are poorly understood.
Furthermore, in contrast to the CesA genes, it has proved difficult to define the functions of the Csl genes. In fact, of the multiple Csl genes in higher plants, only the CslA and CslF groups have been assigned a function.
In accordance with the present invention, it has been revealed that members of the CslH gene family encode (1,3;1,4)-β-
As a result of the identification of CslH nucleotide sequences, and corresponding amino acid sequences that encode (1,3;1,4)-β-
Therefore, in a first aspect, the present invention provides a method for modulating the level of (1,3;1,4)-β-
The “cell” may be any suitable eukaryotic or prokaryotic cell. As such, a “cell” as referred to herein may be a eukaryotic cell including a fungal cell such as a yeast cell or mycelial fungus cell; an animal cell such as a mammalian cell or an insect cell; or a plant cell. Alternatively, the cell may also be a prokaryotic cell such as a bacterial cell including an E. coli cell, or an archaea cell.
In some embodiments, the cell is a plant cell, a vascular plant cell, including a monocotyledonous or dicotyledonous angiosperm plant cell, or a gymnosperm plant cell. In some embodiments the plant is a monocotyledonous plant cell. In some embodiments, the plant is a member of the order Poales. In some embodiments, the monocotyledonous plant cell is a cereal crop plant cell.
As used herein, the term “cereal crop plant” includes members of the Poales (grass family) that produce edible grain for human or animal food. Examples of Poales cereal crop plants which in no way limit the present invention include wheat, rice, maize, millet, sorghum, rye, triticale, oats, barley, teff, wild rice, spelt and the like. However, the term cereal crop plant should also be understood to include a number of non-Poales species that also produce edible grain and are known as the pseudocereals, such as amaranth, buckwheat and quinoa.
In other embodiments, the present invention also contemplates the use of other monocotyledonous plants, such as other non-cereal plants of the Poales, specifically including pasture grasses such as Lolium spp.
As set out above, the present invention is predicated, in part, on modulating the level and/or activity of a CslH-encoded (1,3;1,4)-β-
A “CslH-encoded (1,3;1,4)-β-
In some embodiments, the CslH-encoded (1,3;1,4)-β-
In some embodiments the CslH-encoded (1,3;1,4)-β-
When comparing amino acid sequences, the compared sequences should be compared over a comparison window of at least 100 amino acid residues, at least 200 amino acid residues, at least 400 amino acid residues, at least 800 amino acid residues or over the full length of SEQ ID NO: 2. The comparison window may comprise additions or deletions (i.e. gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms such the BLAST family of programs as, for example, disclosed by Altschul et al. (Nucl. Acids Res. 25: 3389-3402, 1997). A detailed discussion of sequence analysis can be found in Unit 19. 3 of Ausubel et al. (Current Protocols in Molecular Biology, John Wiley & Sons Inc, 1994-1998, Chapter 15, 1998).
Examples of additional CslH-encoded (1,3;1,4)-β-
For example, barley (Hordeum vulgare) orthologs or allelic variants of SEQ ID NO: 2 include, for example, polypeptides comprising the amino acid sequence set forth in SEQ ID NO: 70. Rice (Oryza sativa) orthologs of SEQ ID NO: 2 include, for example, polypeptides comprising the amino acid sequences set forth in any of SEQ ID NO: 4, SEQ ID NO: 6 and SEQ ID NO: 8. Wheat (Triticum aestivum) orthologs of SEQ ID NO: 2 include, for example, polypeptides comprising the amino acid sequences set forth in SEQ ID NO: 75, SEQ ID NO: 76 and SEQ ID NO: 77.
As referred to herein, modulation of the “level” of the CslH-encoded (1,3;1,4)-β-
By “modulating” with regard to the level and/or activity of the CslH-encoded (1,3;1,4)-β-
In some embodiments, the level of (1,3;1,4)-β-
The methods of the present invention contemplate any means known in the art by which the level and/or activity of a CslH-encoded (1,3;1,4)-β-
In some embodiments, the level and/or activity of a (1,3;1,4)-β-
Therefore, in a second aspect, the present invention provides a method for modulating the level and/or activity of a (1,3;1,4)-β-
As used herein, the term “CslH nucleic acid” should be understood to include to a nucleic acid molecule which:
-
- encodes a CslH-encoded (1,3;1,4)-β-
D -glucan synthase as defined herein; and/or - comprises at least 50% nucleotide sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1; and/or
- hybridises to a nucleic acid molecule comprising one or more of the nucleotide sequence set forth in SEQ ID NO: 1 under stringent conditions.
- encodes a CslH-encoded (1,3;1,4)-β-
In some embodiments the CslH nucleic acid comprises at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.5%, at least 96%, at least 96.5%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99% at least 99.5% or 100% sequence identity to SEQ ID NO: 1.
When comparing nucleic acid sequences to SEQ ID NO: 1 to calculate a percentage identity, the compared nucleotide sequences should be compared over a comparison window of at least 300 nucleotide residues, at least 600 nucleotide residues, at least 1200 nucleotide residues, at least 2400 nucleotide residues or over the full length of SEQ ID NO: 1. The comparison window may comprise additions or deletions (ie. gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms such the BLAST family of programs as, for example, disclosed by Altschul et al. (Nucl. Acids Res. 25: 3389-3402, 1997). A detailed discussion of sequence analysis can be found in Unit 19. 3 of Ausubel et al. (“Current Protocols in Molecular Biology” John Wiley & Sons Inc, 1994-1998, Chapter 15, 1998).
As set out above, the CslH nucleic acid may also comprise a nucleic acid that hybridises to a nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO: 1 under stringent conditions. As used herein, “stringent” hybridisation 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 30° C. Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Stringent hybridisation conditions may be low stringency conditions, medium stringency conditions or high stringency conditions. Exemplary low stringency conditions include hybridisation with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C. Exemplary medium stringency conditions include hybridisation in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringency conditions include hybridisation in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C. Optionally, wash buffers may comprise about 0.1% to about 1% SDS. Duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours.
Specificity of hybridisation is also a function of post-hybridization washes, and is influenced by the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the Tm can be approximated from the equation of Meinkoth and Wahl (Anal. Biochem. 138: 267-284, 1984), ie. Tm=81.5° C.+16.6 (log M)+0.41 (% GC)-0.61 (% form)-500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. Tm is reduced by about 1° C. for each 1% of mismatching; thus, Tm, hybridization, and/or wash conditions can be adjusted to hybridize to sequences of different degrees of complementarity. For example, sequences with ≧90% identity can be hybridised by decreasing the Tm by about 10° C. Generally, stringent conditions are selected to be lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength and pH. For example, high stringency conditions can utilize a hybridization and/or wash at, for example, 1, 2, 3, 4 or 5° C. lower than the thermal melting point (Tm); medium stringency conditions can utilize a hybridization and/or wash at, for example, 6, 7, 8, 9, or 10° C. lower than the thermal melting point (Tm); low stringency conditions can utilize a hybridization and/or wash at, for example, 11, 12, 13, 14, 15, or 20° C. lower than the thermal melting point (Tm). Using the equation, hybridization and wash compositions, and desired Tm, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a Tm of less than 45° C. (aqueous solution) or 32° C. (formamide solution), the SSC concentration may be increased so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen (Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, Pt I, Chapter 2, Elsevier, New York, 1993), Ausubel et al., eds. (Current Protocols in Molecular Biology, Chapter 2, Greene Publishing and Wiley-Interscience, New York, 1995) and Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y., 1989).
Examples of additional CslH nucleic acids contemplated by the present invention include nucleic acids having coding regions which are orthologs of SEQ ID NO: 1.
For example, barley (Hordeum vulgare) coding region orthologs or allelic variants of SEQ ID NO: 1 include, for example, nucleic acids comprising the nucleotide sequence set forth in SEQ ID NO: 69. Rice (Oryza sativa) coding region orthologs of SEQ ID NO: 1 include, for example, nucleic acids comprising the nucleotide sequence set forth in any of SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7. Wheat (Triticum aestivum) coding region orthologs of SEQ ID NO: 1 include, for example, nucleic acids comprising the nucleotide sequence set forth in SEQ ID NO: 72, SEQ ID NO: 73 and SEQ ID NO: 74.
The CslH nucleic acids contemplated by the present invention may also comprise one or more non-translated regions such as 3′ and 5′ untranslated regions and/or introns. For example, the CslH nucleic acids contemplated by the present invention may comprise, for example, mRNA sequences, cDNA sequences or genomic nucleotide sequences
In some specific embodiments, the CslH nucleic acid may comprise a genomic nucleotide sequence from an organism which may include one or more non-protein-coding regions and/or one or more introns. Genomic nucleotide sequences which comprise a CslH nucleic acid include, for example:
-
- barley (Hordeum vulgare) CslH genomic nucleotide sequences, for example, as set forth in SEQ ID NO: 9 and/or SEQ ID NO: 71;
- rice (Oryza sativa) CslH genomic nucleotide sequences, for example, as set forth in any one or more of SEQ ID NO: 10, SEQ ID NO: 11 and/or SEQ ID NO: 12; and/or
- wheat (Triticum aestivum) CslH genomic nucleotide sequences, for example, as set forth in any one or more of SEQ ID NO: 78, SEQ ID NO: 79 and/or SEQ ID NO: 80.
As mentioned above, the present invention provides methods for modulating the expression of a CslH nucleic acid in a cell. The present invention contemplates any method by which the expression of a CslH nucleic acid in a cell may be modulated.
The term “modulating” with regard to the expression of the CslH nucleic acid is generally intended to refer to decreasing or increasing the transcription and/or translation of a CslH nucleic acid. By “decreasing” is intended, for example, a 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% reduction in the transcription and/or translation of a CslH nucleic acid. By “increasing” is intended, for example a 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold or greater increase in the transcription and/or translation of a CslH nucleic acid. Modulating also comprises introducing expression of a CslH nucleic acid not normally found in a particular cell; or the substantially complete inhibition (eg. knockout) of expression of a CslH nucleic acid in a cell that normally has such activity.
Methods for modulating the expression of a particular nucleic acid molecule in a cell are known in the art and the present invention contemplates any such method. Exemplary methods for modulating the expression of a CslH nucleic acid include: genetic modification of the cell to upregulate or downregulate endogenous CslH nucleic acid expression; genetic modification by transformation with a CslH nucleic acid; administration of a nucleic acid molecule to the cell which modulates expression of an endogenous CslH nucleic acid in the cell; and the like.
In some embodiments, the expression of a CslH nucleic acid is modulated by genetic modification of the cell. The term “genetically modified”, as used herein, should be understood to include any genetic modification that effects an alteration in the expression of a CslH nucleic acid in the genetically modified cell relative to a non-genetically modified form of the cell. Exemplary types of genetic modification contemplated herein include: random mutagenesis such as transposon, chemical, UV or phage mutagenesis together with selection of mutants which overexpress or underexpress an endogenous CslH nucleic acid; transient or stable introduction of one or more nucleic acid molecules into a cell which direct the expression and/or overexpression of CslH nucleic acid in the cell; site-directed mutagenesis of an endogenous CslH nucleic acid; introduction of one or more nucleic acid molecules which inhibit the expression of an endogenous CslH nucleic acid in the cell, eg. a cosuppression construct or an RNAi construct; and the like.
In one particular embodiment, the genetic modification comprises the introduction of a nucleic acid into a cell of interest.
The nucleic acid may be introduced using any method known in the art which is suitable for the cell type being used, for example, those described in Sambrook and Russell (Molecular Cloning—A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory Press, 2000).
In some embodiments of the invention wherein the cell is a plant cell, suitable methods for introduction of a nucleic acid molecule may include: Agrobacterium-mediated transformation, microprojectile bombardment based transformation methods and direct DNA uptake based methods. Roa-Rodriguez et al. (Agrobacterium-mediated transformation of plants, 3rd Ed. CAMBIA Intellectual Property Resource, Can berra, Australia, 2003) review a wide array of suitable Agrobacterium-mediated plant transformation methods for a wide range of plant species. Microprojectile bombardment may also be used to transform plant tissue and methods for the transformation of plants, particularly cereal plants, and such methods are reviewed by Casas et al. (Plant Breeding Rev. 13: 235-264, 1995). Direct DNA uptake transformation protocols such as protoplast transformation and electroporation are described in detail in Galbraith et al. (eds.), Methods in Cell Biology Vol. 50, Academic Press, San Diego, 1995). In addition to the methods mentioned above, a range of other transformation protocols may also be used. These include infiltration, electroporation of cells and tissues, electroporation of embryos, microinjection, pollen-tube pathway, silicon carbide- and liposome mediated transformation. Methods such as these are reviewed by Rakoczy-Trojanowska (Cell. Mol. Biol. Lett. 7: 849-858, 2002). A range of other plant transformation methods may also be evident to those of skill in the art.
The introduced nucleic acid may be single stranded or double stranded. The nucleic acid may be transcribed into mRNA and translated into a CslH-encoded (1,3;1,4)-β-
In one particular embodiment, the present invention contemplates increasing the level of (1,3;1,4)-β-
By identifying CslH nucleotide sequences which encode (1,3;1,4)-β-
For example, the identification of CslH genes as encoding (1,3;1,4)-β-
-
- insertional mutagenesis of a CslH nucleic acid in a cell, including knockout or knockdown of a CslH nucleic acid in a cell, by homologous recombination with a knockout construct (for an example of targeted gene disruption in plants see Terada et al., Nat. Biotechnol. 20: 1030-1034, 2002);
- post-transcriptional gene silencing (PTGS) or RNAi of a CslH nucleic acid in a cell (for review of PTGS and RNAi see Sharp, Genes Dev. 15(5): 485-490, 2001; and Hannon, Nature 418: 244-51, 2002);
- transformation of a cell with an antisense construct directed against a CslH nucleic acid (for examples of antisense suppression in plants see van der Krol et al., Nature 333: 866-869; van der Krol et al., Bio Techniques 6: 958-967; and van der Krol et al., Gen. Genet. 220: 204-212);
- transformation of a cell with a co-suppression construct directed against a CslH nucleic acid (for an example of co-suppression in plants see van der Krol et al., Plant Cell 2(4): 291-299);
- transformation of a cell with a construct encoding a double stranded RNA directed against a CslH nucleic acid (for an example of dsRNA mediated gene silencing see Waterhouse et al., Proc. Natl. Acad. Sci. USA 95: 13959-13964, 1998);
- transformation of a cell with a construct encoding an siRNA or hairpin RNA directed against a CslH nucleic acid (for an example of siRNA or hairpin RNA mediated gene silencing in plants see Lu et al., Nucl. Acids Res. 32(21): e171; doi:10.1093/nar/gnh170, 2004); and/or
- insertion of a miRNA target sequence such that it is in operable connection with CslH nucleic acid (for an example of miRNA mediated gene silencing see Brown et al., Blood 110(13): 4144-4152, 2007).
The present invention also facilitates the downregulation of a CslH nucleic acid in a cell via the use of synthetic oligonucleotides such as siRNAs or microRNAs directed against a CslH nucleic acid which are administered to a cell (for examples of synthetic siRNA mediated silencing see Caplen et al., Proc. Natl. Acad. Sci. USA 98: 9742-9747, 2001; Elbashir et al., Genes Dev. 15: 188-200, 2001; Elbashir et al., Nature 411: 494-498, 2001; Elbashir et al., EMBO J. 20: 6877-6888, 2001; and Elbashir et al., Methods 26: 199-213, 2002).
In addition to the examples above, the introduced nucleic acid may also comprise a nucleotide sequence which is not directly related to a CslH nucleic acid but, nonetheless, may directly or indirectly modulate the expression of CslH nucleic acid in a cell. Examples include nucleic acid molecules that encode transcription factors or other proteins which promote or suppress the expression of an endogenous CslH nucleic acid molecule in a cell; and other non-translated RNAs which directly or indirectly promote or suppress endogenous CslH-encoded (1,3;1,4)-β-
In order to effect expression of an introduced nucleic acid in a genetically modified cell, where appropriate, the introduced nucleic acid may be operably connected to one or more control sequences. The term “control sequences” should be understood to include any nucleotide sequences which are necessary or advantageous for the transcription, translation and or post-translational modification of the operably connected nucleic acid or the transcript or protein encoded thereby. Each control sequence may be native or foreign to the operably connected nucleic acid. The control sequences may include, but are not limited to, a leader, polyadenylation sequence, propeptide encoding sequence, promoter, enhancer or upstream activating sequence, signal peptide encoding sequence, and transcription terminator. Typically, a control sequence at least includes a promoter.
The term “promoter” as used herein, describes any nucleic acid which confers, activates or enhances expression of a nucleic acid molecule in a cell. Promoters are generally positioned 5′ (upstream) to the genes that they control. In the construction of heterologous promoter/gene combinations, it may be desirable to position the promoter at a distance from the gene transcription start site that is approximately the same as the distance between that promoter and the gene it controls in its natural setting, ie. the gene from which the promoter is derived. As is known in the art, some variation in this distance can be accommodated without loss of promoter function.
A promoter may regulate the expression of an operably connected nucleotide sequence constitutively, or differentially with respect to the cell, tissue, organ or developmental stage at which expression occurs, in response to external stimuli such as physiological stresses, pathogens, or metal ions, amongst others, or in response to one or more transcriptional activators. As such, the promoter used in accordance with the methods of the present invention may include a constitutive promoter, an inducible promoter, a tissue-specific promoter or an activatable promoter.
The present invention contemplates the use of any promoter which is active in a cell of interest. As such, a wide array of promoters which are active in any of bacteria, fungi, animal cells or plant cells would be readily ascertained by one of ordinary skill in the art. However, in some embodiments, plant cells are used. In these embodiments, plant-active constitutive, inducible, tissue-specific or activatable promoters are typically used.
Plant constitutive promoters typically direct expression in nearly all tissues of a plant and are largely independent of environmental and developmental factors. Examples of constitutive promoters that may be used in accordance with the present invention include plant viral derived promoters such as the Cauliflower Mosaic Virus 35S and 19S (CaMV 35S and CaMV 19S) promoters; bacterial plant pathogen derived promoters such as opine promoters derived from Agrobacterium spp., eg. the Agrobacterium-derived nopaline synthase (nos) promoter; and plant-derived promoters such as the rubisco small subunit gene (rbcS) promoter, the plant ubiquitin promoter (Pubi), the rice actin promoter (Pact) and the oat globulin promoter.
“Inducible” promoters include, but are not limited to, chemically inducible promoters and physically inducible promoters. Chemically inducible promoters include promoters which have activity that is regulated by chemical compounds such as alcohols, antibiotics, steroids, metal ions or other compounds. Examples of chemically inducible promoters include: alcohol regulated promoters (eg. see European Patent 637 339); tetracycline regulated promoters (eg. see U.S. Pat. No. 5,851,796 and U.S. Pat. No. 5,464,758); steroid responsive promoters such as glucocorticoid receptor promoters (eg. see U.S. Pat. No. 5,512,483), estrogen receptor promoters (eg. see European Patent Application 1 232 273), ecdysone receptor promoters (eg. see U.S. Pat. No. 6,379,945) and the like; metal-responsive promoters such as metallothionein promoters (eg. see U.S. Pat. No. 4,940,661, U.S. Pat. No. 4,579,821 and U.S. Pat. No. 4,601,978); and pathogenesis related promoters such as chitinase or lysozyme promoters (eg. see U.S. Pat. No. 5,654,414) or PR protein promoters (eg. see U.S. Pat. No. 5,689,044, U.S. Pat. No. 5,789,214, Australian Patent 708850, U.S. Pat. No. 6,429,362).
The inducible promoter may also be a physically regulated promoter which is regulated by non-chemical environmental factors such as temperature (both heat and cold), light and the like. Examples of physically regulated promoters include heat shock promoters (eg. see U.S. Pat. No. 5,447,858, Australian Patent 732872, Canadian Patent Application 1324097); cold inducible promoters (eg. see U.S. Pat. No. 6,479,260, U.S. Pat. No. 6,084,08, U.S. Pat. No. 6,184,443 and U.S. Pat. No. 5,847,102); light inducible promoters (eg. see U.S. Pat. No. 5,750,385 and Canadian Patent 132 1563); light repressible promoters (eg. see New Zealand Patent 508103 and U.S. Pat. No. 5,639,952).
“Tissue specific promoters” include promoters which are preferentially or specifically expressed in one or more specific cells, tissues or organs in an organism and/or one or more developmental stages of the organism. It should be understood that a tissue specific promoter may, in some cases, also be inducible.
Examples of plant tissue specific promoters include: root specific promoters such as those described in US Patent Application 2001047525; fruit specific promoters including ovary specific and receptacle tissue specific promoters such as those described in European Patent 316 441, U.S. Pat. No. 5,753,475 and European Patent Application 973 922; and seed specific promoters such as those described in Australian Patent 612326 and European Patent application 0 781 849 and Australian Patent 746032.
In some embodiments, the tissue specific promoter is a seed and/or grain specific promoter. Exemplary seed or grain specific promoters include puroindoline-b gene promoters (for example see Digeon et al., Plant Mol. Biol. 39: 1101-1112, 1999); Pbf gene promoters (for example see Mena et al., Plant J. 16: 53-62, 1998); GS1-2 gene promoters (for example see Muhitch et al., Plant Sci. 163: 865-872, 2002); glutelin or Gt1 gene promoters (for example see Okita et al., J. Biol. Chem. 264: 12573-12581, 1989; Zheng et al., Plant J. 4: 357-366, 1993; Sindhu et al., Plant Sci. 130: 189-196, 1997; Nandi et al., Plant Sci. 163: 713-722, 2002); Hor2-4 gene promoters (for example see Knudsen and Müller, Planta 195: 330-336, 1991; Patel et al., Mol. Breeding 6: 113-123, 2000; Wong et al., Proc. Natl. Acad. Sci. USA 99: 16325-16330, 2002); lipoxygenase 1 gene promoters (for example see Rouster et al., Plant J. 15: 435-440, 1998); Chi26 gene promoters (for example see Leah et al., Plant J. 6: 579-589, 1994); Glu-D1-1 gene promoters (for example see Lamacchia et al., J. Exp. Bot. 52: 243-250, 2001; Zhang et al., Theor. Appl. Genet. 106: 1139-1146, 2003); Hor3-1 gene promoters (for example see Sörensen et al., Mol. Gen. Genet. 250: 750-760, 1996; Horvath et al., Proc. Natl. Acad. Sci. USA 97: 1914-1919, 2000) and Waxy (Wx) gene promoters (for example see Yao et al., Acta Phytophysiol. Sin. 22: 431-436, 1996; Terada et al., Plant Cell Physiol. 41: 881-888, 2000; Liu et al., Transgenic Res. 12: 71-82, 2003). In one specific embodiment, the seed specific promoter is an endosperm specific promoter.
The promoter may also be a promoter that is activatable by one or more defined transcriptional activators, referred to herein as an “activatable promoter”. For example, the activatable promoter may comprise a minimal promoter operably connected to an Upstream Activating Sequence (UAS), which comprises, inter alia, a DNA binding site for one or more transcriptional activators.
As referred to herein the term “minimal promoter” should be understood to include any promoter that incorporates at least an RNA polymerase binding site and, preferably a TATA box and transcription initiation site and/or one or more CAAT boxes. When the cell is a plant cell, the minimal promoter may be derived from, for example, the Cauliflower Mosaic Virus 35S (CaMV 35S) promoter. The CaMV 35S derived minimal promoter may comprise, for example, a sequence that corresponds to positions −90 to +1 (the transcription initiation site) of the CaMV 35S promoter (also referred to as a −90 CaMV 35S minimal promoter), −60 to +1 of the CaMV 35S promoter (also referred to as a −60 CaMV 35S minimal promoter) or −45 to +1 of the CaMV 35S promoter (also referred to as a −45 CaMV 35S minimal promoter).
As set out above, the activatable promoter may comprise a minimal promoter fused to an Upstream Activating Sequence (UAS). The UAS may be any sequence that can bind a transcriptional activator to activate the minimal promoter. Exemplary transcriptional activators include, for example: yeast derived transcription activators such as Gal4, Pdr1, Gcn4 and Ace1; the viral derived transcription activator, VP16; Hap1 (Hach et al., J Biol Chem 278: 248-254, 2000); Gaf1 (Hoe et al., Gene 215(2): 319-328, 1998); E2F (Albani et al., J Biol Chem 275: 19258-19267, 2000); HAND2 (Dai and Cserjesi, J Biol Chem 277: 12604-12612, 2002); NRF-1 and EWG (Herzig et al., J Cell Sci 113: 4263-4273, 2000); P/CAF (Itoh et al., Nucl Acids Res 28: 4291-4298, 2000); MafA (Kataoka et al., J Biol Chem 277: 49903-49910, 2002); human activating transcription factor 4 (Liang and Hai, J Biol Chem 272: 24088-24095, 1997); Bcl10 (Liu et al., Biochem Biophys Res Comm 320(1): 1-6, 2004); CREB-H (Omori et al., Nucl Acids Res 29: 2154-2162, 2001); ARR1 and ARR2 (Sakai et al., Plant J 24(6): 703-711, 2000); Fos (Szuts and Bienz, Proc Natl Acad Sci USA 97: 5351-5356, 2000); HSF4 (Tanabe et al., J Biol Chem 274: 27845-27856, 1999); MAML1 (Wu et al., Nat Genet 26: 484-489, 2000).
In some embodiments, the UAS comprises a nucleotide sequence that is able to bind to at least the DNA-binding domain of the GAL4 transcriptional activator. UAS sequences, which can bind transcriptional activators that comprise at least the GAL4 DNA binding domain, are referred to herein as UASc. In a particular embodiment, the UASc comprises the sequence 5′-CGGAGTACTGTCCTCCGAG-3′ or a functional homolog thereof.
As referred to herein, a “functional homolog” of the UASc sequence should be understood to refer to any nucleotide sequence which can bind at least the GAL4 DNA binding domain and which may comprise a nucleotide sequence having at least 50% identity, at least 65% identity, at least 80% identity or at least 90% identity with the UASc nucleotide sequence.
The UAS sequence in the activatable promoter may comprise a plurality of tandem repeats of a DNA binding domain target sequence. For example, in its native state, UASc comprises four tandem repeats of the DNA binding domain target sequence. As such, the term “plurality” as used herein with regard to the number of tandem repeats of a DNA binding domain target sequence should be understood to include at least 2, at least 3 or at least 4 tandem repeats.
As mentioned above, the control sequences may also include a terminator. The term “terminator” refers to a DNA sequence at the end of a transcriptional unit which signals termination of transcription. Terminators are 3′-non-translated DNA sequences generally containing a polyadenylation signal, which facilitates the addition of polyadenylate sequences to the 3′-end of a primary transcript. As with promoter sequences, the terminator may be any terminator sequence which is operable in the cells, tissues or organs in which it is intended to be used. Examples of suitable terminator sequences which may be useful in plant cells include: the nopaline synthase (nos) terminator, the CaMV 35S terminator, the octopine synthase (ocs) terminator, potato proteinase inhibitor gene (pin) terminators, such as the pinII and pinIII terminators and the like.
Modulating the level of (1,3;1,4)-β-
For example, (1,3;1,4)-β-
Soluble cereal (1,3;1,4)-β-
However, cereal (1,3;1,4)-β-
Humans and monogastric animals produce no enzymes that degrade (1,3;1,4)-β-
Therefore, in another embodiment, the present invention may be applied to increasing the dietary fibre content of an edible plant or edible plant part, by increasing the level of (1,3;1,4)-β-
(1,3;1,4)-β-
The present invention also facilitates the production of (1,3;1,4)-β-
A vast array of recombinant expression systems that may be used to express a CslH nucleic acid are known in the art. Exemplary recombinant expression systems include: bacterial expression systems such as E. coli expression systems (reviewed in Baneyx, Curr. Opin. Biotechnol. 10: 411-421, 1999; eg. see also Gene expression in recombinant microorganisms, Smith (Ed.), Marcel Dekker, Inc. New York, 1994; and Protein Expression Technologies: Current Status and Future Trends, Baneyx (Ed.), Chapters 2 and 3, Horizon Bioscience, Norwich, UK, 2004), Bacillus spp. expression systems (eg. see Protein Expression Technologies: Current Status and Future Trends, supra, chapter 4) and Streptomyces spp. expression systems (eg. see Practical Streptomyces Genetics, Kieser et al., (Eds.), Chapter 17, John Innes Foundation, Norwich, UK, 2000); fungal expression systems including yeast expression systems such as Saccharomyces spp., Schizosaccharomyces pombe, Hansenula polymorpha and Pichia spp. expression systems and filamentous fungi expression systems (eg. see Protein Expression Technologies: Current Status and Future Trends, supra, chapters 5, 6 and 7; Buckholz and Gleeson, Bio/Technology 9(11): 1067-1072, 1991; Cregg et al., Mol. Biotechnol. 16(1): 23-52, 2000; Cereghino and Cregg, FEMS Microbiology Reviews 24: 45-66, 2000; Cregg et al., Bio/Technology 11: 905-910, 1993); mammalian cell expression systems including Chinese Hamster Ovary (CHO) cell based expression systems (eg. see Protein Expression Technologies: Current Status and Future Trends, supra, chapter 9); insect cell cultures including baculovirus expression systems (eg. see Protein Expression Technologies: Current Status and Future Trends, supra, chapter 8; Kost and Condreay, Curr. Opin. Biotechnol. 10: 428-433, 1999; Baculovirus Expression Vectors: A Laboratory Manual WH Freeman & Co., New York, 1992; and The Baculovirus Expression System: A Laboratory Manual, Chapman & Hall, London, 1992); Plant cell expression systems such as tobacco, soybean, rice and tomato cell expression systems (eg. see review of Hellwig et al., Nat Biotechnol 22: 1415-1422, 2004); and the like.
Therefore, in a third aspect, the present invention provides a method for producing (1,3;1,4)-β-
In some embodiments, the cell is a cell from a recombinant expression system as hereinbefore defined.
In a fourth aspect, the present invention also provides (1,3;1,4)-β-
In a fifth aspect, the present invention also provides a cell comprising:
-
- a modulated level and/or activity of CslH-encoded (1,3;1,4)-β-
D -glucan synthase relative to a wild type cell of the same taxon; and/or - modulated expression of a CslH nucleic acid relative to a wild type cell of the same taxon.
- a modulated level and/or activity of CslH-encoded (1,3;1,4)-β-
In some embodiments, the cell further comprises a modulated level of (1,3;1,4)-β-
In some embodiments, the cell of the fifth aspect of the invention is produced according to the methods of the first or second aspects of the present invention as described herein. In further embodiments, the cell is a plant cell, a monocot plant cell, a Poales plant cell and/or a cereal crop plant cell.
Furthermore, in a sixth aspect, the present invention provides a multicellular structure comprising one or more cells according to the fifth aspect of the invention.
As referred to herein, a “multicellular structure” includes any aggregation of one or more cells. As such, the term “multicellular structure” specifically encompasses tissues, organs, whole organisms and parts thereof. Furthermore, a multicellular structure should also be understood to encompass multicellular aggregations of cultured cells such as colonies, plant calli, suspension cultures and the like.
As mentioned above, in some embodiments of the invention, the cell is a plant cell and as such, the present invention includes a whole plant, plant tissue, plant organ, plant part, plant reproductive material or cultured plant tissue, comprising one or more plant cells according to the sixth aspect of the invention.
In another embodiment, the present invention provides a cereal crop plant comprising one or more cells according to the fifth aspect of the invention.
In a particular embodiment, the present invention provides cereal grain comprising one or more cells according to the fifth aspect of the invention.
Therefore, in a seventh aspect, the present invention provides a cereal grain comprising a modulated level of (1,3;1,4)-β-
In some embodiments, the grain may have an increased level of (1,3;1,4)-β-
In some embodiments wherein the grain is a wheat grain, the wheat grain comprises level of (1,3;1,4)-β-
In an eighth aspect, the present invention also provides flour comprising:
-
- flour produced by the milling of the grain of the seventh aspect of the invention; and
- optionally, flour produced by the milling of one or more other grains.
As such, the flour produced by the milling of the grain of the seventh aspect of the invention may comprise, for example approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% by weight of the flour of the ninth aspect of the invention.
As referred to herein “milling” contemplates any method known in the art for milling grain, such as those described by Brennan et al. (Manual of Flour and Husk Milling, Brennan et al. (Eds.), AgriMedia, ISBN: 3-86037-277-7).
In some embodiments, the flour produced by the milling of the grain of the seventh aspect of the invention used in the flour comprises an increased level of (1,3;1,4)-β-
The “flour produced by the milling of one or more other grains” may be flour produced by milling grain derived from any cereal plant, as hereinbefore defined. This component of the flour of the eighth aspect of the invention may, for example, comprise 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% by weight.
In some embodiments, the flour produced by the milling of one or more other grains is wheat flour and, therefore, the flour of the eighth aspect of the invention may be particularly suitable for producing bread, cakes, biscuits and the like.
As set out above, the present invention is predicated, in part, on the identification and isolation of CslH nucleotide sequences and CslH amino acid sequences that encode (1,3;1,4)-β-
Therefore, in a ninth aspect, the present invention provides an isolated CslH nucleic acid as hereinbefore defined, or a complement, reverse complement or fragment thereof.
In the present invention, “isolated” refers to material removed from its original environment (e.g., the natural environment if it is naturally occurring), and thus is altered “by the hand of man” from its natural state. For example, an isolated polynucleotide could be part of a vector or a composition of matter, or could be contained within a cell, and still be isolated because that vector, composition of matter, or particular cell is not the original environment of the polynucleotide. An “isolated” nucleic acid molecule should also be understood to include a synthetic nucleic acid molecule, including those produced by chemical synthesis using known methods in the art or by in-vitro amplification (eg. polymerase chain reaction and the like).
The isolated nucleic acid molecules of the present invention may comprise any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. For example, the isolated nucleic acid molecules of the invention may comprise single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and/or double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, the isolated nucleic acid molecules may comprise of triple-stranded regions comprising RNA or DNA or both RNA and DNA. The isolated nucleic acid molecules may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically, or metabolically modified forms.
As set out above, the present invention also provides fragments of a nucleotide sequence. “Fragments” of a nucleotide sequence should be at least 15, 20, 30, 40, 50, 100, 150, 200, 250, 300, 325, 350, 375, 400, 450, 500, 550, or 600 nucleotides (nt) in length. These fragments have numerous uses that include, but are not limited to, diagnostic probes and primers. Of course, larger fragments, such as those of 601-3000 nt in length are also useful according to the present invention as are fragments corresponding to most, if not all, of a CslH nucleic acid.
In some embodiments, the fragment may comprise a functional fragment of a CslH nucleic acid. That is, the polynucleotide fragments of the invention may encode a polypeptide having (1,3;1,4)-β-
In a tenth aspect, the present invention provides a genetic construct or vector comprising an isolated nucleic acid molecule of the ninth aspect of the invention.
The vector or construct may further comprise one or more of: an origin of replication for one or more hosts; a selectable marker gene which is active in one or more hosts; or one or more control sequences which enable transcription of the isolated nucleic acid molecule in a cell.
“Selectable marker genes” include any nucleotide sequences which, when expressed by a cell, confer a phenotype on the cell that facilitates the identification and/or selection of these transformed cells. A range of nucleotide sequences encoding suitable selectable markers are known in the art. Exemplary nucleotide sequences that encode selectable markers include: antibiotic resistance genes such as ampicillin-resistance genes, tetracycline-resistance genes, kanamycin-resistance genes, the AURI-C gene which confers resistance to the antibiotic aureobasidin A, neomycin phosphotransferase genes (eg. nptI and nptII) and hygromycin phosphotransferase genes (eg. hpt); herbicide resistance genes including glufosinate, phosphinothricin or bialaphos resistance genes such as phosphinothricin acetyl transferase encoding genes (eg. bar), glyphosate resistance genes including 3-enoyl pyruvyl shikimate 5-phosphate synthase encoding genes (eg. aroA), bromyxnil resistance genes including bromyxnil nitrilase encoding genes, sulfonamide resistance genes including dihydropterate synthase encoding genes (eg. sul) and sulfonylurea resistance genes including acetolactate synthase encoding genes; enzyme-encoding reporter genes such as GUS and chloramphenicolacetyltransferase (CAT) encoding genes; fluorescent reporter genes such as the green fluorescent protein-encoding gene; and luminescence-based reporter genes such as the luciferase gene, amongst others.
Furthermore, it should be noted that the selectable marker gene may be a distinct open reading frame in the construct or may be expressed as a fusion protein with the CslH-encoded (1,3;1,4)-β-
The tenth aspect of the invention extends to all genetic constructs essentially as described herein, which include further nucleotide sequences intended for the maintenance and/or replication of the genetic construct in prokaryotes or eukaryotes and/or the integration of the genetic construct or a part thereof into the genome of a eukaryotic or prokaryotic cell.
In some embodiments, the vector or construct is adapted to be at least partially transferred into a plant cell via Agrobacterium-mediated transformation. Accordingly, the vector or construct may comprise left and/or right T-DNA border sequences.
Suitable T-DNA border sequences would be readily ascertained by one of skill in the art. However, the term “T-DNA border sequences” may include substantially homologous and substantially directly repeated nucleotide sequences that delimit a nucleic acid molecule that is transferred from an Agrobacterium sp. cell into a plant cell susceptible to Agrobacterium-mediated transformation. By way of example, reference is made to the paper of Peralta and Ream (Proc. Natl. Acad. Sci. USA, 82(15): 5112-5116, 1985) and the review of Gelvin (Microbiology and Molecular Biology Reviews, 67(1): 16-37, 2003).
Although in some embodiments, the vector or construct is adapted to be transferred into a plant via Agrobacterium-mediated transformation, the present invention also contemplates any suitable modifications to the genetic construct which facilitate bacterial mediated insertion into a plant cell via bacteria other than Agrobacterium sp., for example, as described in Broothaerts et al. (Nature 433: 629-633, 2005).
Those skilled in the art will be aware of how to produce the constructs described herein and of the requirements for obtaining the expression thereof, when so desired, in a specific cell or cell-type under the conditions desired. In particular, it will be known to those skilled in the art that the genetic manipulations required to perform the present invention may require the propagation of a genetic construct described herein or a derivative thereof in a prokaryotic cell such as an E. coli cell or a plant cell or an animal cell. Exemplary methods for cloning nucleic acid molecules are described in Sambrook et al. (2000, supra)
In an eleventh aspect, the present invention provides a cell comprising the isolated nucleic acid molecule of the ninth aspect of the invention or genetic construct of the tenth aspect of the invention.
The isolated nucleic acid molecule of the tenth or eleventh aspects of the invention or genetic construct of the twelfth aspect of the invention may be introduced into a cell via any means known in the art.
The isolated nucleic acid molecule or construct referred to above may be maintained in the cell as a DNA molecule, as part of an episome (eg. a plasmid, cosmid, artificial chromosome or the like) or it may be integrated into the genomic DNA of the cell.
As used herein, the term “genomic DNA” should be understood in its broadest context to include any and all DNA that makes up the genetic complement of a cell. As such, the genomic DNA of a cell should be understood to include chromosomes, mitochondrial DNA, plastid DNA, chloroplast DNA, endogenous plasmid DNA and the like. As such, the term “genomically integrated” contemplates chromosomal integration, mitochondrial DNA integration, plastid DNA integration, chloroplast DNA integration, endogenous plasmid integration, and the like.
The isolated nucleic acid molecule may be operably connected to, inter alia, a control sequence and/or a promoter such that the cell may express the isolated nucleic acid molecule.
The cell may be any prokaryotic or eukaryotic cell. As such, the cell may be a prokaryotic cell such as a bacterial cell including an E. coli cell or an Agrobacterium spp. cell, or an archaea cell. The cell may also be a eukaryotic cell including a fungal cell such as a yeast cell or mycelial fungus cell; an animal cell such as a mammalian cell or an insect cell; or a plant cell. In a particular embodiment, the cell is a plant cell. In some embodiments, the plant cell is a monocot plant cell, a Poales plant cell, or a cereal crop plant cell.
In a twelfth aspect, the present invention provides a multicellular structure, as hereinbefore defined, comprising one or more of the cells of the eleventh aspect of the invention.
As mentioned above, in some embodiments, the cell is a plant cell and as such, the present invention should be understood to specifically include a whole plant, plant tissue, plant organ, plant part, plant reproductive material, or cultured plant tissue, comprising one or more cells of the eleventh aspect of the invention.
In a further embodiment, the present invention provides a monocot plant, a Poales plant or a cereal crop plant or part thereof, comprising one or more cells of the eleventh aspect of the invention.
In some embodiments, the present invention provides cereal grain comprising one or more cells of the eleventh aspect of the invention.
As set out above, the present invention also provides amino acid sequences for CslH-encoded (1,3;1,4)-β-
The isolated polypeptides may comprise of amino acids joined to each other by peptide bonds or modified peptide bonds, ie., peptide isosteres, and may contain amino acids other than the 20 gene-encoded amino acids. The isolated polypeptides of the present invention may be modified by either natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art.
Modifications can occur anywhere in the isolated polypeptide, including the peptide backbone, the amino acid side-chains and/or the termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given isolated polypeptide. Also, an isolated polypeptide of the present invention may contain many types of modifications.
The polypeptides may be branched, for example, as a result of ubiquitination, and/or they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from post-translation natural processes or may be made by synthetic methods.
Modifications include acetylation, acylation, ADP-ribosylation, amidation, biotinylation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, PEGylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. (See, for instance, Proteins—Structure And Molecular Properties 2nd Ed., Creighton (ed.), W. H. Freeman and Company, New York, 1993); Posttranslational Covalent Modification Of Proteins, Johnson (Ed.), Academic Press, New York, 1983; Seifter et al., Meth Enzymol 182: 626-646, 1990); Rattan et al., Ann NY Acad Sci 663: 48-62, 1992).
As set out above, the present invention also provides fragments of isolated polypeptides. Polypeptide fragments may be “free-standing” or comprised within a larger polypeptide of which the fragment forms a part or region.
The polypeptide fragments can be at least 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acids in length.
In some embodiments, the fragment is a functional fragment and thus comprises (1,3;1,4)-β-
A CslH-encoded (1,3;1,4)-β-
A polypeptide comprising one or more CslH-encoded (1,3;1,4)-β-
The isolated CslH-encoded (1,3;1,4)-β-
Such antibodies are useful, inter alia, in the detection and localization of (1,3;1,4)-β-
Accordingly, in a fourteenth aspect, the present invention provides an antibody or an epitope binding fragment thereof, raised against an isolated CslH-encoded (1,3;1,4)-β-
The antibodies of the present invention include, but are not limited to, polyclonal, monoclonal, multispecific, chimeric antibodies, single chain antibodies, Fab fragments, F(ab′) fragments, fragments produced by a Fab expression library and epitope-binding fragments of any of the above.
The term “antibody”, as used herein, refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site that immunospecifically binds an antigen. The immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.
The antibodies of the present invention may be monospecific, bispecific, trispecific, or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of a polypeptide of the present invention or may be specific for both a polypeptide of the present invention as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material. For example, see PCT publications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt et al., J. Immunol. 147: 60-69, 1991; U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; and Kostelny et al. J. Immunol. 148: 1547-1553, 1992).
In some embodiments, the antibodies of the present invention may act as agonists or antagonists of CslH-encoded (1,3;1,4)-β-
The term “antibody”, as used herein, should be understood to encompass derivatives that are modified, eg. by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from binding to a CslH-encoded (1,3;1,4)-β-
Antibodies may be generated using methods known in the art.
For example, if in vivo immunization is used, animals may be immunized with free peptide; however, anti-peptide antibody titer may be boosted by coupling of the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid. For example, peptides containing cysteine residues may be coupled to a carrier using a linker such as maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to carriers using a more general linking agent such as glutaraldehyde.
Animals such as rabbits, rats and mice may be immunized with either free or carrier-coupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 micrograms of peptide or carrier protein and Freund's adjuvant. Several booster injections may be needed, for example, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody which can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface. The titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, for instance, by adsorption to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.
Polyclonal antibodies to a CslH-encoded (1,3;1,4)-β-
As another example, monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed., 1988) and Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas (Elsevier, NY, 1981). The term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology. The term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art. For example, mice can be immunized with a polypeptide of the invention or a cell expressing such peptide. Once an immune response is detected, e.g., antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well-known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC. Hybridomas are selected and cloned by limited dilution. The hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding a polypeptide of the invention. Ascites fluid, which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.
Antibody fragments which recognize one or more CslH-encoded (1,3;1,4)-β-
The antibodies of the present invention can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In a particular embodiment, such phage can be utilized to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labelled antigen or antigen bound or captured to a solid surface or bead. Phages used in these methods are typically filamentous phage including fd and M13 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein.
Examples of phage display methods include those disclosed by Brinkman et al. (J. Immunol. Methods 182: 41-50, 1995), Ames et al. (J. Immunol. Methods 184: 177-186, 1995), Kettleborough et al. (Eur. J. Immunol. 24: 952-958, 1994), Persic et al. (Gene 187: 9-18, 1997), Burton et al. (Advances in Immunology 57: 191-280, 1994); PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108.
After phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria. For example, techniques to recombinantly produce Fab, Fab′ and F(ab′)2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication WO 92/22324; Mullinax et al. (BioTechniques 12(6): 864-869, 1992); and Sawai et al. (AJRI 34:26-34, 1995); and Better et al. (Science 240: 1041-1043, 1988).
Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al. (Methods in Enzymology 203: 46-88, 1991); Shu et al. (Proc. Natl. Acad. Sci. USA 90: 7995-7999, 1993); and Skerra et al. (Science 240: 1038-1040, 1988).
The present invention is further described by the following non-limiting examples:
Candidate CSLH genes in barley were initially identified by querying online EST databases, such as the discontinued Stanford cell wall website, NCBI, HarvEST, GrainGenes, Barley Gene Index and BarleyBase, with rice CSLH sequences. All CSLH-related ESTs from barley could be aligned into a single contiguous sequence of ˜1,500 bp that included the entire 3′ untranslated region (UTR) and a region encoding the COOH-terminal 488 (of an expected ˜750) amino acid residues of the protein (Table 2). This gene was designated HvCslH1. Screening of a barley BAC library with HvCslH1-derived probes identified several genomic clones all containing HvCslH1, from which the missing 5′ end was obtained (data not shown). A 2,430 bp HvCslH1 cDNA fragment was PCR-amplified, contains a single 2,256 bp ORF, and encodes a protein with a predicted MW of 82.6 kDa and a pI of 7.0 (
A BAC library screening was employed to obtain a complete set of full-length HvCslH family members. BAC filters containing 6.5 equivalents of the barley genome (cv. Morex) were screened and three clearly positive clones identified (data not shown). When a blot of BAC DNA from these clones digested with Hind III was probed, the same three clones, 3-5-10, 3-7-3 and 3-7-8, were verified as being positive. The digestion pattern of BACs 3-5-10 and 3-7-8 appeared identical and many bands were common to BAC 3-7-3, indicating that all 3 BACs cover identical or very similar regions of the barley genome. When a genomic DNA blot was hybridised with the same probe, single bands were observed in lanes digested with Hind III, Eco RI or Eco RV, corroborating the BAC digestion results. As all HvCslH ESTs are also derived from a single gene (Table 2), these data strongly suggest that there is only one CSLH gene in the barley genome.
An adaptor primer PCR method (Siebert et al., Nucl Acids Res 23: 1087-1088, 1995) was used to identify the 5′ end of HvCslH1. DNA was isolated from BACs 3-5-10 and 3-7-3, digested with a range of restriction enzymes producing blunt-ended DNA fragments to which adaptors were ligated. Nested PCR was then performed with adaptor- and HvCslH1-specific primers (Table 3) in order to amplify fragments containing the 5′ end of the gene. Amplification of BAC 3-7-3 DNA digested with Nru I using primers AP2 and H1R6 successfully amplified a 1.3 kbp fragment that contained all but ˜20 amino acids of the N-terminal sequence. Direct sequencing of BAC 3-7-3 DNA with the H1R10 primer, an antisense primer designed to anneal near the 5′ end of the 1.3 kb fragment, enabled the remainder of the open reading frame plus 748 bp of upstream sequence to be identified. As predicted from earlier results, the sequence obtained from BAC 3-5-10 was identical to BAC 3-7-3, confirming that there is only one CSLH gene within the barley genome.
For heterologous expression in Arabidopsis, the HvCslH1 ORF was cloned into the Gateway-enabled binary vector pGWB15 (Nakagawa et al., J Biosci Bioeng 104: 34-41, 2007; FIG. 15), which placed HvCslH1 under the control of the CaMV 35S promoter and added a 3×HA epitope tag to the encoded protein's NH2-terminal end (
Immuno-EM was used to determine whether the walls of the transgenic Arabidopsis plants accumulated detectable levels of β-glucan. Thin sections of mature leaf pieces from self-pollinated progeny of lines 8, 11, 16, 24 and 6 (T2 generation) were probed with a monoclonal antibody specific for β-glucan (Meikle et al., Plant J 5: 1-9, 1994), followed by detection using a secondary antibody conjugated to 18 nm gold particles. Gold particles were clearly evident in walls of the HA-tagged HvCslH1 positive lines 8, 11 and 16 (
To provide biochemical confirmation of the presence of β-glucan in transgenic Arabidopsis walls and to examine the fine structure of the nascent β-glucan, leaf and/or stem material was pooled from the self-pollinated T3 and T4 progeny of lines derived from plants 8, 11 and 16. These lines were homozygous for the HvCslH1 transgene. Because β-glucan was found to accumulate with plant age, samples were taken when plants were in senescence. Walls were prepared and digested with a (1,3:1,4)-β-glucan-specific endo-hydrolase and the released oligosaccharides profiled by HPAEC and MALDI-TOF MS. (1,3;1,4)-β-D-Glucan endo-hydrolase specifically hydrolyses (1,4)-β-glucosidic linkages when these linkages are on the reducing-end side of a (1,3)-β-D-glucosyl residue. The action of this enzyme yields a series of oligosaccharides with different degrees of polymerization (DP). The diagnostic oligosaccharides in this series are the trisaccharide G4G3GR and the tetrasaccharide G4G4G3GR (where G is β-D-glucopyranose, 3 and 4 indicate (1,3) and (1,4) linkages, respectively, and GR refers to the reducing terminal residue). Variable quantities of G4G3GR and G4G4G3GR were released when walls prepared from leaf or leaf and stem from lines 8 and 11 and two independent lines derived from plant 16 (lines 16-1 and 16-2) were treated with (1,3;1,4)-β-D-glucan endo-hydrolase (
Sections of high pressure-frozen leaves from line 11 were incubated with the gold-labelled anti-HA antibody to determine the sub-cellular location of HvCslH1. Labelling was seen in the endoplasmic reticulum and in Golgi-derived vesicles but not in the plasma membrane (
The levels of HvCslH1 transcripts in various barley tissues were determined using quantitative RT-PCR (QPCR). The gene-specific primers are presented in Table 4.
The highest levels of HvCslH1 transcripts were in leaf tip, where cells are no longer actively growing and less β-glucan accumulates (
HvCslH1 transcript levels were also investigated in more detail in a 24-day developing endosperm series (
The data presented here indicate that the product of HvCslH1, a member of the grass-specific CSLH gene family, mediates β-glucan biosynthesis in Arabidopsis. Barley appears to have only a single CSLH gene based on EST database analyses, genomic DNA blot analysis and BAC library screening. EST analyses of other grasses such as bread wheat, Lolium multiflorum, Festuca arundinacae and Brachypodium distachon (all subfamily Pooideae) have one identified CSLH gene, similar to barley, whereas maize, sorghum and sugar cane (all subfamily Panicoideae), like rice (subfamily Ehrhartoideae), appear to have multiple CSLH genes. When an epitope-tagged version of the HvCslH1 cDNA was heterologously expressed in Arabidopsis, three of four plant lines in which protein was detected accumulated a polysaccharide in their walls that was recognized by a β-glucan-specific monoclonal antibody. When isolated walls of the transgenic lines were digested with a specific (1,3;1,4)-β-D-glucan endo-hydrolase, the characteristic trisaccharide (G4G3GR) and tetrasaccharide (G4G4G3GR) were detected at ratios similar to those found in β-glucans from barley endosperm, demonstrating that the walls from the transgenic Arabidopsis lines contained β-glucan. Furthermore, epitope-tagged HvCslH1 was found in the endoplasmic reticulum and in Golgi-derived vesicles in cells of transgenic plants. The morphological phenotype of the transgenic Arabidopsis lines that expressed HvCslH1 appeared identical to wild-type plants.
Although the overall proportion of (1,3)- and (1,4)-β-glucosyl linkages and the ratios of the G4G3GR and G4G4G3GR products from (1,3;1,4)-β-D-glucan endo-hydrolase digestion of walls derived from plant line 16-1 was similar to those observed in β-glucans isolated from barley tissues, one unusual feature that was observed was that the major oligosaccharide released by (1,3;1,4)-β-D-glucan endo-hydrolase from the walls of line 16-2 was laminaribiose (G3GR;
In barley, HvCslH1 was most highly transcribed in leaf tips, a tissue comprising fully mature cells. There is no evidence to indicate coordinate transcription of HvCslH1 and any of the barley CSLFs, suggesting that their encoded products are not components of a protein complex. HvCslH1 transcription, for example, was not high in elongating cells such as the coleoptile or developing endosperm, which in barley are the tissues where β-glucan is characteristically accumulated. Although usually found in primary cell walls of vegetative tissues where it is implicated in the control of cell expansion and possibly as a temporary store of glucose that can be mobilized as an energy source in the dark, β-glucan has also been found in the lignified cell walls of xylem tracheary elements and sclerenchyma fibres, where immuno-EM using the antibody to β-glucan shows labeling in both the middle lamella region (primary wall) and secondary wall of sclerenchyma cells. Because in situ PCR showing transcription of the HvCslH1 gene in the leaf was restricted to cells such as interfascicular sclerenchymal fibre and xylem cells, we suggest that a major role of this gene is in β-glucan synthesis during secondary wall development, although we cannot exclude a role in primary wall β-glucan synthesis elsewhere in the plant.
Regardless of how the fine structures of β-glucans are generated, it is clear that the CSLHs can mediate the synthesis of β-glucan in Arabidopsis, a finding that has implications for our understanding of how this polysaccharide is synthesised. Any mechanism(s) being considered for the assembly of β-glucan must account for the synthesis of the predominant cellotriosyl and cellotetraosyl units, the random linking of these (1,4)-β-units together by single (1,3)-β-linkages and the means by which the molar ratio of tri- to tetra-saccharide units is regulated. At least two glycosyltransferase activities might act in concert: one that processively adds (1,4)-β-linked glucose residues to assemble the tri- and tetra-saccharides and the other that adds single (1,3)-β-linkages. Based on our current knowledge of polysaccharide synthases several mechanisms are hypothetically possible. The simplest explanation is that the one polypeptide is responsible for the synthesis of both glucosidic linkage types. Our transgenic experiments indicate that CSLH proteins are independently able to make a β-glucan and could therefore conceivably make both types of β-linkages. The CSLH family is classified by the Carbohydrate Active Enzymes (CAZy) database as members of glycosyltransferase family 2 (GT2) (http://www.cazy.org; Coutinho et al., J Mol Biol 328: 307-317, 2003), a family that includes enzymes capable of independently catalyzing the synthesis of either (1,3)-β- or (1,4)-β-linkages but also examples of bifunctional enzymes, i.e. enzymes that can synthesize two types of glycosidic linkages. For example, hyaluronan synthases (HAS) synthesize a repeating disaccharide of (1,4)-β-glucuronic acid-(1,3)-β-N-acetylglucosamine units and both transferase activities reside in the one polypeptide. In mouse HAS1, the region that includes the D,D,D,QXXRW motif is responsible for both these activities. The active site of the CSLHs, also containing the D,D,D,QXXRW motif, might be similarly bifunctional. Another possibility is that the CSLHs synthesise only one type of glucosidic linkage with another glucosyltransferase, common to monocots and dicots, responsible for synthesis of a second linkage.
EXAMPLE 6 Materials and Methods Binary Vector Construction and Plant TransformationThe HvCslH1 ORF was amplified from barley cv. Schooner mature leaf tip cDNA with Herculase® (Stratagene) using primers HvH1TOPOf and HvH1TOPOr (Table 3) and the PCR product cloned into pENTR/D-TOPO (Invitrogen). Using the manufacturer's protocol (Invitrogen), an LR reaction was used to clone the cDNA into the destination vector pGWB15 containing an NH2-terminal 3×HA tag (Nakagawa et al., J Biosci Bioeng 104: 34-41, 2007) and the predicted sequence confirmed by DNA sequencing. The HvCslH1::pGBW15 construct was transferred from Escherichia coli into Agrobacterium tumefaciens strain AGL1 via triparental mating using the helper plasmid pRK2013. Arabidopsis thaliana Col-0 plants were transformed using the floral dip method (Clough and Bent, Plant J 16: 735-743, 1998).
RNA Blot AnalysisSamples of ˜10 μg total RNA extracted from mature rosette leaves of T1 plants using TRIzol® (Invitrogen) were prepared and separated on a 1% w/v agarose-formaldehyde gel (Farrell, RNA methodologies: A laboratory guide for isolation and characterization, Academic Press, Inc., San Diego, 1993). RNA was transferred to Hybond™ N+ membranes, pre-hybridised and hybridised according to the method outlined in the Gene Images CDP-Star detection module (Amersham-Biosciences). A HvCslH1 fragment amplified with primers H1F2 and HvH1TOPOr (Table 3) was labeled using the Gene Images Random Prime labeling module (Amersham) following the manufacturer's protocol and used as the probe.
Quantitative PCR AnalysisRNA extractions, cDNA syntheses and QPCR were carried out as described in Burton et al. (Science 311, 1940-1942, 2006; Plant Physiol 134, 224-236, 2004) with the modifications listed in Burton et al. (Plant Physiol 146, 1821-1833, 2008). The primer sequences of the barley control genes are listed in Table 4.
In Situ PCRIn situ PCRs were conducted according to the method of Koltai & Bird (Plant Physiol 123: 1203-1212, 2000) with the following modifications. After tissue sectioning, genomic DNA was removed by treatment for 6 h at 37° C. in 1× DNase buffer and 4 U RNase-free DNase (Promega). cDNA synthesis was carried out using Thermoscript™ RT (Invitrogen) except that the RNase H step was omitted and a gene-specific primer (1 μg, Table 3) used for reverse transcription. PCRs were carried out in a final volume of 50 μL containing 1×PCR buffer, 200 μm dNTPs (Promega), 0.2 nmol digoxigenin-11-dUTP (Roche), 2 mM MgCl2, 200 ng of each primer and 2 U Taq DNA polymerase (Invitrogen). Cycling parameters were as follows: initial denaturation at 96° C. for 2 min, then 40 cycles of 94° C. for 30 sec, 59° C. for 30 sec, 72° C. for 1 min. Sections were then washed, incubated with 1.5 U alkaline phosphatase-conjugated anti-digoxigenin Fab fragments (Roche) and developed for 10-20 min as outlined by Koltai & Bird (2000, supra). For negative control sections, reverse transcriptase was omitted and all the Hv 18S rRNA primers included to check whether there was any amplification from genomic DNA.
Preparation of Mixed Microsomal MembranesT1 seed of HvCslH1 transgenic plants was collected and ˜100 seeds sown onto 1×MS agar media containing 50 mg/L kanamycin (Sigma). After 3 weeks, kanamycin-resistant seedlings were pooled, frozen in liquid N2 and ground at 4° C. in a mortar and pestle containing homogenising buffer (50 mM NaPO4 buffer, pH 7.5, 0.5 M sucrose, 20 mM KCl, 10 mM DTT, 0.2 mM PMSF, 83 μL plant protease inhibitor cocktail (Sigma, P9599)). Homogenate was filtered through a 50 μM mesh and the S/N centrifuged at 6,000×g for 10 min at 4° C. The S/N was decanted and centrifuged at 50,000×g for 30 min at 4° C. in 4.5 ml ultracentrifuge tubes (Beckmann). The 50,000×g S/N was decanted and the pellet resuspended in 10 mM Tris-MES buffer, pH 7.5 using a glass-teflon homogenizer. The resuspended pellet was diluted to 4.5 mL with Tri-MES buffer and centrifuged at 100,000×g for 1 h at 4° C. The pellet was resuspended in 0.25 M sucrose, 10 mM Tris-MES buffer, pH 7.5, as outlined above. Protein concentration was measured using Bradford assay reagent (BioRad) using bovine serum albumin as the standard.
Western BlottingSamples of membrane protein (30 μg) were incubated at 60° C. for 20-60 min in 200 mM dithiothreitol and sample buffer (37.5 mM Tris-HCl, pH 7.0, 10% glycerol, 3% sodium dodecylsulphate (SDS), 0.025% bromophenol blue) to give an SDS:protein ratio of 1.5 mg SDS to 30 μg protein before loading onto an 8% SDS-PAGE gel. After electrophoresis, gels were blotted onto nitrocellulose (OSMONIC™ Nitropure 22 μm) in Towbin buffer (25 mM Tris base, 192 mM glycine, 20% methanol) containing 0.05% SDS at 100 V for 90 min at 4° C. Membranes were then blocked overnight in Tris-buffered saline (TBS; 20 mM Tris base, 150 mM NaCl) containing 3% w/v milk powder before incubation for 1 h at RT in rat anti-HA polyclonal antibody (Roche) diluted 1:1000 in TBS containing 1% BSA. Membranes were washed 3× in TBS containing 0.05% SDS (TBST), then incubated in anti-rat IgG HRP-conjugated antibody (Dako) diluted 1:1000 in TBS containing 3% w/v nonfat milk powder. Membranes were washed 3× in TBST before signal was detected with the SuperSignal® West Pico chemiluminescent substrate (Pierce).
Immuno-Electron MicroscopyArabidopsis tissues were fixed and labeled with anti-(1,3;1,4)-β-
Alcohol insoluble residue (AIR) was prepared by grinding plant material in liquid N2 using a mortar and pestle. Five volumes of 80% ethanol was added to the homogenate prior to mixing by rotation for 1 h at 4° C. After centrifugation at 3,400×g for 5 min, the supernatant was removed and the residue was refluxed twice at RT in 80% ethanol for 1 h, followed by refluxing in 50% ethanol twice for 1 h. The ethanol-soluble fraction was removed and the AIR was washed once in 100% ethanol prior to drying at 40° C. under vacuum.
(1,3;1,4)-β-AIR (100 mg, prepared as described above) was incubated in 5 mL 20 mM NaPO4 buffer, pH 6.5 for 2 h at 50° C. with continuous mixing in an incubator with shaking at 200 rpm. After 2 h, the suspension was centrifuged (3,400×g, 5 min) and the supernatant (S/N) removed. Another 5 mL of buffer was added and the incubation and centrifugation repeated. The S/N from this second incubation was used as the no enzyme negative control. The pelleted AIR was resuspended in 5 mL NaPO4 buffer to which 100 μl (1,3;1,4)-β-D-glucan endo-hydrolase (McCleary et al., J Inst Brew 91: 285-295, 1985) was added. The mixture was incubated for 2 h at 50° C. with continuous mixing after which the S/N was collected as the (1,3;1,4)-β-D-glucan endo-hydrolase-released oligosaccharides. The negative control and (1,3;1,4)-β-D-glucan endo-hydrolase-treated S/Ns were desalted on a graphitised carbon cartridge as described by Packer et al. (Glycoconj J 15: 737-747, 1998) and dried.
HPAEC AnalysisThe dried (1,3;1,4)-β-D-glucan endo-hydrolase-released oligosaccharides were dissolved in 100 μL Milli H2O and 20 μL injected onto a CarboPac PA1 column (Dionex) equilibrated with 50 mM NaOAc in 0.2 M NaOH using a Dionex BioLC ICS 300 system (Dionex) equipped with a pulsed amperometric detector (PAD) and autosampler. Oligosaccharides were eluted at 1 mL/min with a linear gradient of NaOAc from 50 mM in 0.2M NaOH to 350 mM in 0.2 M NaOH over 15 min. Laminaribiose (Seigaku), maltose and cellobiose (both from Sigma) were run as standards.
MALDI-TOF MS AnalysisAliquots (30 μL) of the remaining (1,3;1,4)-β-
CSLH ESTs were obtained by querying public databases including the now discontinued Stanford Cell Wall website, NCBI (http://www.ncbi.nlm.nih.gov/), HarvEST (http://harvest.ucr.edu/), GrainGenes (http://wheat.pw.usda.gov/GG2/index.shtml), Barley Gene Index (http://compbio.dfci.harvard.edu/tgi/plant.html) and BarleyBase (www.barleybase.org) using the BLAST search tool (Altschul et al., Nucl Acids Res 25: 3389-3402, 1997). Sequences were assembled into contigs using either Sequencer™ 3.0 (GeneCodes) or ContigExpress, a module of Vector NTI® Advance 9.1.0 (Invitrogen). DNA or protein sequences were aligned using ClustalX (Thompson et al., Nucl Acids Res 24: 4876-4882, 1997). Phylogenetic analysis was carried out using the in-built neighbour joining algorithm and tree robustness assessed using 1000 bootstrapped replicates. Sequence similarities were calculated using MatGat 2.02 (http://bitincka.com/ledion/matgat/) (Campanella et al., BMC Bioinformatics 4: 29, 2003). Transmembrane domains were predicted using the suite of programs described in ARAMEMNON (http://aramemnon.botanik.uni-koeln.de) (Schwacke et al., Plant Physiol 131: 16-26, 2003). Motifs predicting post-translational modifications were identified using the programs listed in ExPasy under the Tools menu (http://www.expasy.org/tools/#pattern). Protein parameters were calculated using ProtParam at ExPasy (http://www.expasy.org/cgi-bin/protparam).
Barley BAC ScreeningBAC filters containing 6.5 equivalents of the barley genome from the non-Yd2 cv. Morex (Clemson University Genomics Institute, CUGI) were blocked for 6 h at 65° C. in prehybidisation solution (0.53 M NaPO4 buffer pH 7.2, 7.5% w/v SDS, 1 mM EDTA, 11 μg/ml salmon sperm DNA). The radiolabeled cDNA and gDNA fragment amplified with primers H1F1 and H1R1 or H1R5 (Table 3) was added and incubated for 24 h at 65° C. Filters were washed 3× with 2×SSC, 0.1% SDS at RT. Final washes were done with 1×SSC, 0.1% SDS. Filters were exposed to X-ray film for 2 d. Positive BAC clones were identified and ordered as directed on the CUGI website (http://www.genome.clemson.edu). Clones were streaked onto LB agar containing 25 μg/ml chloramphenical and grown overnight at 37° C. Colonies for each clone were picked, placed on gridded nylon membranes resting on LB agar containing 25 μg/ml chloramphenicol and incubated overnight at 37° C. DNA was fixed to the membrane and denatured by placing on filter paper soaked in 0.4 M NaOH for 20 min, then neutralized by placing on filter paper soaked in neutralizing solution (1.5 M NaCl, 0.5 M Tris-HCl pH 7.2, 1 mM EDTA). Membranes were then washed 3× in 2×SSC, 0.1% SDS and hybridized using standard conditions (Sambrook et al., Molecular cloning: a laboratory manual, Cold Spring Harbour Laboratory Press, New York, 1989).
BAC DNA IsolationPositive clones were cultured overnight in LB broth containing 25 μg/ml chloramphenicol at 37° C. Cells were pelleted by centrifugation (12,000×g, 3 min) and the pellet resuspended in 90 μL TES buffer (25 mM Tris-HCl pH 8.0, 10 mM EDTA, 15% w/v sucrose). An aliquot (180 μL) of lysis solution (0.2 M NaOH, 1% SDS) was added and mixed gently, followed by 135 μL 3 M NaOAc pH 4.6. The chromosomal DNA was pelleted by centrifugation (12,000×g, 15 min). The S/N was collected and 2 μL RNase A (10 mg/mL) added and incubated for 1 h at 37° C. A 400 μL aliquot of Tris-saturated phenol-chloroform (1:1 ratio) was added and the samples centrifuged again (12,000×g, 5 min). The S/N was collected and BAC DNA precipitated using 2-3 volumes chilled 95% ethanol for 10 min at RT. The BAC DNA was pelleted by centrifugation (15,000×g, 15 min), washed in 70% ethanol, resuspended in 20-50 μL TE and stored at 4° C.
Genome WalkingThe adaptor ligation method of Siebert et al. (Nucl Acids Res 23: 1087-1088, 1995) was used to amplify fragments of genomic DNA upstream of known CSLH EST sequence. Restriction enzymes used to digest barley genomic DNA were Eco RV, Nru I, Pvu II, Sca I or Ssp I. Primary PCR reactions were conducted in 25 μL volumes containing 2 μL ligated DNA (1:10 dilution), 1×PCR buffer, 2 mM MgCl2, 100 ng each of adaptor primer AP1 and antisense primer H1R7 (Table 3), 0.4 mM dNTPs and 1 unit Taq polymerase (Invitrogen). Cycle parameters were as follows: 96° C. for 2 min then 40 cycles of 94° C. for 30 sec, 59° C. for 30 sec, 72° C. for 1 min, and a final step at 72° C. for 7 min. A secondary PCR reaction was conducted with 1 μL of the primary PCR using 100 ng each of adaptor primer AP2 and the nested primer H1R6. Reaction composition and cycle parameters were the same as above except that an annealing temperature of 61° C. was used.
BAC SequencingFor sequencing, between 0.5 and 1 μg of isolated BAC DNA was combined with 5 pmol primer and 1× Big Dye Terminator v 3.1 (BDT) mix (Applied Biosystems, USA) in a final volume of 20 μL. Cycle parameters were as follows: 96° C. for 15 min, then 65 cycles of 96° C. for 10 sec, 55° C. for 10 sec and 60° C. for 4 min. DNA was precipitated with 0.1 vol 3M NaOAc pH 5.2 and 2.5 vol 95% ethanol on ice for 10 min, then pelleted by spinning at 12,000×g for 30 min. The pellet was rinsed with 70% ethanol, dried and sent to AGRF (Brisbane, Australia) for sequencing.
Mapping of HvCslH1Genetic mapping was done using a Sloop×Halcyon doubled haploid (DH) mapping population of 60 lines (Read et al., Aust J Agric Res 54: 1145-1153, 2003). Using standard methods of DNA blot hybridization (Sambrook et al., 1989, supra) a HvCslH1 probe PCR-amplified using primers H1F1 and H1R5 (Table 3) was hybridized to membranes containing parental line genomic DNA digested with one of six restriction enzymes (Bam HI, Dra I, Eco RI, Eco RV, Hind III, Xba I). The dihybrid population was then digested with enzymes that gave a clear polymorphism (Dra I). Polymorphisms were scored and HvCslH1 map location determined using the ‘find best location’ function of MapManager QT version 0.30 (Manly et al., Mamm Genome 12: 930-932, 2001). Map locations were correlated with QTL data using resources available at http://www.barleyworld.org/.
Arabidopsis Growth ConditionsArabidopsis seeds were surface-sterilized in a sterilization solution (sodium hypochlorite (2% available chlorine), drop of Tween-20) for 15 min then rinsed 4× with sterile MilliQ water.
Surface-sterilized seed was spread onto 85×25 mm Petri dishes containing 50 mL of sterile 1×MS medium (4.33 g/L Murashige and Skoog basal salts (Phytotechnology Laboratories), 2% w/v sucrose, 1% w/v bactoagar). For selection of transformants, 50 mg/L kanamycin (Sigma) was added to the medium. Plates were placed in a cold room for 3-5 days at 4° C. to synchronize germination. Cold-stratified plates were then transferred into controlled environment growth cabinets (Thermoline L+M model TPG 1260 TO-5×400, Smithfield, NSW, Australia) with day and night temperatures of 23° C. and 17° C., respectively. The average light intensity at rosette leaf level was ˜70 μE m−2 sec−1 supplied by 3-foot fluorescent tubes (Sylvania Standard F30W/133-T8 Cool White) during a 16 h light cycle. After 3 weeks on MS plates, individual plantlets were transferred into hydrated 42 mm diameter Jiffy pellets. Nine rows of six pellets were arranged in trays with three trays being housed on each 2×3.5-foot wire rack shelf. Relative humidity was measured to be between 60 and 70%. Plants were watered with tap water supplemented with Peter's Professional™ General Purpose plant fertilizer (Scotts Australia) by sub-irrigation every 2-3 days.
Genomic DNA Extraction and PCR Analysis of Arabidopsis TransgenicsDNA was extracted from a single Arabidopsis leaf according to the method described in Edwards et al. (Nucl Acids Res 19: 1349, 1991). A 1 μL aliquot of genomic DNA was used as template in PCR screens of transgenic plants using primers H1F2 and HvCslH1TOPOr (Table 3) with the following cycling regime: 94° C. for 2 min followed by 35 cycles of 94° C. for 20 sec, 57° C. for 30 sec, 72° C. for 30 sec.
EXAMPLE 7 Alignment of CslH DNA and Amino Acid Sequences from Rice and BarleyAn alignment of the DNA and amino acid sequences for the CslH sequences in both rice and barley was performed to calculate the percent identity and similarity between the sequences, the results of which are shown in
Multiple sequence alignments and phylogenetic tree generation was performed using the ClustalX program as described by Thompson et al. (Nucl Acids Res 25: 4876-4882, 1997). The protein alignment and resultant phylogenetic tree are shown in
Two transgenic Arabidopsis lines, 15-8 and 15-11, in which the tagged HvCslH1 protein was detected using an anti-HA antibody, were chosen to genetically cross with two other transgenic Arabidopsis lines containing OsCslF2, H37 and H17-4, as described by Burton et al. (Science 311: 1940-1942, 2006). It was thought that by expressing the HvCslH1 and OsCSLF2 proteins in the same cell types, higher levels of (1,3;1,4)-β-
All four of the parental lines were confirmed to contain (1,3;1,4)-β-
The progeny of each cross were sown in soil and their genotypes determined by PCR using leaf genomic DNA as template and HvCslH1-specific primers and, in a separate reaction, OsCslF2-specific primers. Mature leaves were fixed, embedded, sectioned and labeled with (1,3;1,4)-β-
A full length cDNA sequence of the CslH1 gene was isolated from barley cultivar Himalaya using a combination of barley EST sequences, PCR from cDNA using primers based on the rice CslH1 gene sequence (LOC_Os10g20090) and 5′RACE.
The 2333 bp consensus sequence designated HvCslH1(Him) (SEQ ID NO: 69) is shown in
Oligonucleotide primers SJ91 and SJ85 were designed from the 5′ and 3′ ends of the consensus sequence and used to amplify a 3203 bp DNA fragment from genomic DNA designated HvCslH1gHim (SEQ ID NO: 71) in
Alignment of the barley cDNA sequence and genomic sequences indicated that the CslH gene has eight small (approximately 100 bp) introns each flanked by the consensus GT . . . AG splice donor/acceptor sites (
A wheat homolog of CslH1 was identified in the TIGR database as TC255929. Three classes of sequences made up this tentative consensus as exemplified by ESTs CJ614392, CJ609729 and CJ721204. PCR primers were designed from the barley sequence surrounding the ATG initiation codon (SJ163) and from the consensus sequence of all three EST types at the 3′ end (SJ164) and used to amplify a full length genomic fragment from wheat cultivar Chinese Spring. Two sequence types were identified and designated TaCslH1-1 (SEQ ID NO: 78) and TaCslH1-2 (SEQ ID NO: 79). The third homeolog designated TaCslH1-3 (SEQ ID NO: 80) was isolated using primers SJ204 and SJ164 as described in more detail in materials and methods.
Comparison with the barley sequences indicated that the intron-exon junctions were conserved in all three genes (
The predicted coding region sequences of the three wheat CslH1 genes (SEQ ID NO: 72, SEQ ID NO: 73 and SEQ ID NO: 74) each encode a polypeptide of 752 amino acids (SEQ ID NO: 75, SEQ ID NO: 76 and SEQ ID NO: 77).
The DNA coding sequences and amino acids sequences of the barley and wheat CSLH1 genes were aligned using the muscle alignment program and the percentage identity and similarity was calculated using a PAM250 matrix. A table showing the percentage identity and similarity is show in
As shown in
Expression of the CslH1 gene was examined by semi quantitative (RT-PCR and gel electrophoresis) and quantitative (real time PCR) methods.
The coleoptile is a good tissue to examine expression of genes related to beta glucan biosynthesis since the levels of beta glucan increase as the coleoptile grows and then decline after growth has stopped. The CslH1 gene shows maximum expression only after growth has ceased and is high in the oldest tissues (6-8 days old, as shown in FIG. 19A/B).
Other tissues were also examined. In developing leaf, the CslH1 gene shows differential and maximum expression in the oldest tissue at the tip of the leaf (
In barley endosperm tissue, CslH1 gene expression peaked around 4 days post anthesis and then increased during later stages to reach a maximum at 28 days (
There was a large difference in CslH1 gene expression in wheat where expression peaked at 4 days post anthesis after which expression was very low. These results were confirmed by real time PCR which showed that at 28 days post anthesis, the CslH gene was expressed about 10 fold higher levels in barley than in wheat (
Transgenic wheat plants were generated by biolistics transformation with the full length genomic HvCslH1 (cv. Himalaya) gene under control of the glutenin promoter such that expression should only occur in endosperm tissues (
Most of the lines expressed the barley CslH gene at several hundred fold higher levels than the controls with line 9, 10, 12 and 14 showing the highest expression (greater than one thousand fold higher).
At maturity, single grains from were analysed for beta glucan content and a summary of the results are shown in Table 6:
The PCR negative lines all had the lowest beta glucan contents averaging 0.69% of grain weight, whereas grain from the PCR positive lines had an increased average beta glucan content of 0.97%. The last column of Table 6 shows the maximum beta glucan content of any single grain from a given line—the highest PCR negative line was 1.0% (and most grains were much lower than this) but several of the PCR positive lines had grains with significantly increased beta glucan levels with line 9 and line 10 (the highest expressers) having grains with up to 1.9% beta glucan. These levels of beta glucan have never been seen before in wheat.
The heads from these T0 plants contain T1 seed which are segregating for the transgene. If the DNA is inserted at a single locus a ratio of three transgenic to one wild type seed should be observed.
It is expected that further increases in beta glucan will be seen in these grains when the lines are made homozygous and gene dosage increases.
EXAMPLE 12 Materials and Methods for Examples 9 to 11 Plant MaterialBarley (Hordeum vulgare) cultivar Himalaya and wheat (Triticum aestivum) cultivar Chinese Spring, Westonia and Bob White26, were grown under standard glasshouse conditions.
Primer SequencesThe primer sequences referred to in Examples 9 to 11 and this example are shown below in Table 7:
DNA, RNA Isolation and cDNA Synthesis
Plant DNA was isolated from fully expanded leaf tissue using a CTAB based method (Murray and Thompson, Nucleic Acids Res. 8: 4321-4325, 1980). Total RNA was isolated from leaf and coleoptile tissues using an RNAeasy kit from Qiagen. RNA was isolated from developing endosperm using a phenol SDS method and LiCl precipitation (Clarke et al., Functional and Integrative Genomics 8, 211-221, 2007). RNA was treated with DNAse using a “DNA-free” kit from Ambion and then cDNA was synthesised using SuperscriptIII reverse transcriptase according to the manufacturer's instructions (Clontech).
Cloning of CslH GenesThe methods for cloning CslH genes were similar to those described in the cloning and characterisation of CslF genes (Burton et al., Plant Physiol 146: 1821-1833, 2008). A 1.8 kb tentative consensus sequence (TC140327) of a barley homolog of the rice Cellulose synthase like H1 gene (LOC_Os10g20090) was identified in the TIGR database. PCR primer pairs (SJ27-SJ73 and SJ72-SJ75) were designed based on the rice CslH1 sequence and used to amplify sequences from cDNA. The 5′ end of the gene was then amplified by 5′RACE using a SMART cDNA library and nested CslH1 primers SJ28 and SJ79 according to the manufacturer's instructions (Clontech).
A full length genomic clone was isolated by amplification with primers SJ91 and SJ85 and Phusion Taq polymerase (Finnzymes) according to the manufacturers recommend cycling conditions (denature 30 sec at 98° C. followed by 35 cycles of 98° C. for 5 sec, 63° C. for 7 sec and 72° C. for 3 min) and cloned into the pCRBluntII TOPO cloning vector (Invitrogen).
Wheat CslH genomic clones were isolated by PCR with Phusion polymerase from the cultivar Chinese Spring using primers SJ163 and SJ164 and an annealing temperature of 70° C. A genome walking kit was used according to the manufacturers instructions (Clontech) to obtain sequences extending upstream of the coding region of all three wheat CslH homeologs from the variety Bob White (data not shown). A primer (SJ204) was designed that was specific to the third homeolog and used with SJ164 to isolate the third full length genomic clone. It was confirmed that the predicted exon/intron boundaries could be spliced correctly by sequencing cDNA fragments (data not shown).
Expression Analysis of CslH Gene in Wheat and Barley by RT-PCR Total RNA was isolated from sections of the first leaf of a 7 day old plant, from dark grown coleoptiles of different ages, and from developing grain collected at 4 day intervals post anthesis (DPA), DNAse treated and reverse transcribed with Superscript III according to the manufacturer's instructions (Invitrogen). PCR reactions were performed using HotStarTaq (Qiagen). The cDNA was diluted and used in PCR reactions at a level equivalent to 1 ng of original RNA per microlitre. For semi-quantitative RT-PCR, CslH1 primers SJ72 and SJ74, for the CslF genes, primer pairs were as follows; (CslF6; SJ107-SJ82), (CslF4; SJ94-SJ95), (CslF9; SJ97-SJ93), (CslF3; SJ44-SJ38), (CslF8; SJ96-SJ37). An annealing temperature of 59° C. was used. Test amplifications were performed to ensure that the amplification was not saturated (approx 32-35 cycles except tubulin 24 cycles) and the products were analysed by ethidium bromide staining after agarose gel electrophoresis. Real time PCR was performed on triplicate samples on a Rotorgene 6000 machine (Corbett Life Sciences, AU) using HotStarTaq (Qiagen), SybrGreen and primers SJ183 and SJ164 and an annealing temperature of 60° C. Relative expression levels were calculated using the machine software with wheat 0 dpa samples as the comparator (set to one). The Ct value of this sample was 25.5 cycles. For analysis of transgenic grain at 15 dpa, the relative expression values were normalised against tubulin and compared to the lowest expression line (H1-13).
Expression Analysis of CslH Gene in Barley by Q-PCRHvCslH1 transcript was measured in developing coleoptile 0.5 to 7 days post germination. HvCslH1 transcript was shown to accumulate only after the completion of the elongation phase and the emergence of the leaf. Highest levels of expression were seen at 7 days when the coleoptile is senescing (twisting and shrinking) (Gibeaut et al., Planta 221:729-738, 2005).
Production of Transgenic Wheat Plants Overexpressing the Barley CslH Gene in EndospermThe full length barley cv. Himalaya genomic CslH sequence (SEQ ID NO: 71) was amplified using primers SJ91 and SJ85, was inserted as an EcoRI fragment between a 1.9 kb fragment of the high molecular weight glutenin Bx17 promoter and the nopaline synthase terminator (
Bob White 26 wheat plants were transformed using the biolistics method (Pellegrineschi et al., Genome 45: 421-430, 2002) with 50 mg/L G418 as the selection agent. The HvCslH expression vector (pZLBx17HvgH1 and a second plasmid with the CaMV 35S promoter driving expression of the NPTII selectable marker (pCMSTLSneo,
Transgenic plants were screened for the presence of the transgene using young leaf tissue and the RedExtractnAmp™ kit from Sigma with primers SJ244 and SJ79.
At anthesis (emergence of the anthers and shedding of pollen) heads were tagged to enable grain to be sampled at approximately 15 dpa. Three grains from a head were pooled, RNA extracted and reverse transcribed and levels of transgene expression were analysed by real time PCR using primers SJ183 and SJ85. Expression levels were normalised against alpha tubulin (primers TUB and TUB2F) and finally expressed as a ratio compared to the lowest expresser.
Flour from mature single grains was analysed for beta glucan content using a scaled down version of the lichenase enzymatic method (AACC Method 32-33, Megazyme assay kit, McCleary and Glennie-Holmes, J. Inst Brewing 91: 285-295, 1985). Beta glucan contents are expressed as a percentage (w/w) of the milled whole grain flour.
EXAMPLE 13 Overexpression of the Barley CslH Gene in Barley Cv. Golden PromiseThe full-length coding region of the barley CslH cDNA (SEQ ID NO: 1) was transferred into two Gateway-enabled barley transformation vectors. The vector pRB474 contains the oat globulin promoter (Vickers et al., Plant Mol Biol 62: 195-214, 2006) which provides endosperm specific expression and the vector pMDC32 (Curtis and Grossniklaus, Plant Physiol. 133: 462-9, 2003) contains a double 35S promoter which drives constitutive expression in all plant tissues.
Barley TransformationThe vectors were transferred into Agrobacterium tumefaciens and immature scutella of the barley cultivar Golden Promise were transformed using established protocols to produce two populations of transgenic plants. Insertion of the transgene was confirmed by Southern blotting. Plants 236-1 to 236-18 contain the barley CslH gene driven by the oat globulin promoter. Plants 237-1 and -2 contain the barley CslH gene driven by the 35S promoter. Plants 208-2, -3, -5 and -7 are control plants and are transgenic for the empty vector pRB474 carrying the oat globulin promoter only.
Transcript AnalysisLeaf and developing grain samples, from 7 and 14 days after pollination (DAP) were collected from the 236 plants. Total RNA was extracted using TRIzol reagent (Invitrogen) following a standard protocol and cDNA was synthesized according to Burton et al., (Plant Physiol 146: 1821-1833, 2008). Quantitative real-time PCR (QPCR) was carried out according to Burton et al. (2008, supra). The transcript levels of the CslH gene were compared in the endosperm of the transgenic grain to wild type endosperm levels which are generally very low.
As shown in
The T1 seed from the transgenic plants were collected. A sample of the bulked T1 grain from each individual plant was ground to flour and the amount of beta-glucan present was assayed using Megazyme method (described supra). The data from each plant are presented as the mean value of two replicates and the amount of beta-glucan as a percentage of grain weight is shown in Table 8, below:
The empty vector control lines (208) have a (1,3;1,4)-β-
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to, or indicated in this specification, individually or collectively, and any and all combinations of any two or more of the steps or features.
Also, it must be noted that, as used herein, the singular forms “a”, “an” and “the” include plural aspects unless the context already dictates otherwise. Thus, for example, reference to “a transgene” includes a single transgene as well as two or more transgenes; “a plant cell” includes a single cell as well as two or more cells; and so forth.
Claims
1. A method for modulating the level of (1,3;1,4)-β-D-glucan produced by a plant or fungal cell, the method comprising modulating the level and/or activity of a CslH-encoded (1,3;1,4)-β-D-glucan synthase in the cell, wherein the level and/or activity of the a CslH-encoded (1,3;1,4)-β-D-glucan synthase is modulated by modulating the expression of a CslH nucleic acid in the cell, and wherein modulating the expression of a CslH nucleic acid in the cell results in modulation of the level of (1,3;1,4)-β-D-glucan produced by the cell compared to a wild-type cell of the same taxon, and wherein the CslH nucleic acid comprises:
- (i) a nucleotide sequence set forth in SEQ ID NO: 72;
- (ii) a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 75; or
- (iii) a nucleotide sequence encoding an amino acid sequence which is at least 50% identical to the amino acid sequence set forth in SEQ ID NO: 75.
2. The method of claim 1 wherein the cell is a plant cell.
3. The method of claim 2 wherein the cell is a monocot plant cell.
4. The method of claim 2 wherein the cell is a cereal crop plant cell.
5. A plant or fungal cell comprising any one or more of:
- a modulated level and/or activity of CslH-encoded (1,3;1,4)-β-D-glucan synthase relative to a wild type cell of the same taxon; and/or
- modulated expression of a CslH nucleic acid relative to a wild type cell of the same taxon,
- wherein the cell comprises a modulated level of (1,3;1,4)-β-D-glucan relative to a wild-type cell of the same taxon,
- wherein the CslH nucleic acid comprises: (i) a nucleotide sequence set forth in SEQ ID NO: 72; (ii) a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 75; or (iii) a nucleotide sequence encoding an amino acid sequence which is at least 50% identical to the amino acid sequence set forth in SEQ ID NO: 75.
6. The cell of claim 5 wherein the cell is produced according to the method of claim 1.
7. The cell of claim 5 wherein the cell is a plant cell.
8. The cell of claim 7 wherein the cell is a monocot plant cell.
9. The cell of claim 7 wherein the cell is a cereal crop plant cell.
10. A multicellular structure comprising one or more cells according to claim 5.
11. The multicellular structure of claim 10 wherein the multicellular structure is selected from the list consisting of a whole plant, a plant tissue, a plant organ, a plant part, plant reproductive material or cultured plant tissue.
12. The multicellular structure of claim 10 wherein the multicellular structure comprises a cereal crop plant or a tissue, organ or part thereof.
13. The multicellular structure of claim 12 wherein the multicellular structure comprises a cereal grain.
14. The multicellular structure of claim 11 wherein the multicellular structure comprises a cell having modulated dietary fibre content relative to a wild type cell of the same taxon.
15. The multicellular structure of claim 14 wherein the multicellular structure comprises a cell having an increased level of (1,3;1,4)-β-D-glucan relative to a wild type cell of the same taxon and an increased dietary fibre content relative to a wild type cell of the same taxon.
Type: Application
Filed: Nov 27, 2013
Publication Date: Jun 12, 2014
Applicants: The University Of Melbourne (Victoria), Grains Research & Development Corporation (Australian Capital Territory), The University Of Adelaide (South Australia), Commonwealth Scientific And Industrial Research Organisation (Australian Capital Territory)
Inventors: Monika Susanne DOBLIN (Victoria), Filomena Angela PETTOLINO (Victoria), Antony BACIC (Victoria), Stephen Alan JOBLING (Australian Capital Territory), Geoffrey Bruce FINCHER (South Australia), Rachel Anita BURTON (South Australia)
Application Number: 14/092,216
International Classification: C12N 15/82 (20060101);
