# Patent

## Title
Manipulation of flavonoid biosynthesis in plants

## Abstract

The present invention relates to nucleic acids and nucleic acid fragments encoding amino acid sequences for flavonoid biosynthetic enzymes in plants, and the use thereof for the modification of flavonoid biosynthesis in plants. More particularly, the flavonoid biosynthetic enzyme is selected from the group consisting of chalcone isomerase (CHI), chalcone synthase (CHS), chalcone reductase (CHR), dihydroflavonol 4-reductase (DFR), leucoanthocyanidin reductase (LCR), flavonoid 3′, 5′ hydrolase (F3′5′H), flavanone 3-hydroxylase (F3H), flavonoid 3′-hydroxylase (F3′H), phenylalanine ammonia-olyase (PAL) and vestitone reductase (VR), and functionally active fragments and variants thereof.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of International patent application PCT/AU2002/001345, filed Oct. 4, 2002, which claims the benefit of Australian patent application PR8113, filed Oct. 5, 2001.
The present invention relates to nucleic acids and nucleic acid fragments encoding amino acid sequences for flavonoid biosynthetic enzymes in plants, and the use thereof for the modification of flavonoid biosynthesis in plants.
BACKGROUND OF THE INVENTION
Flavonoids constitute a relatively diverse family of aromatic molecules that are derived from phenylalanine and malonyl-coenzyme A (CoA, via the fatty acid pathway). These compounds include six major subgroups that are found in most higher plants: the chalcones, flavones, flavonols, flavandiols, anthocyanins and condensed tannins (or proanthocyanidins). A seventh group, the aurones, is widespread, but not ubiquitous.
Some plant species also synthesize specialized forms of flavonoids, such as the isoflavonoids that are found in legumes and a small number of non-legume plants. Similarly, sorghum, maize and gloxinia are among the few species known to synthesize 3-deoxyanthocyanins (or phlobaphenes in the polymerised form). The stilbenes which are closely related to flavonoids, are synthesised by another group of unrelated species that includes grape, peanut and pine.
Besides providing pigmentation to flowers, fruits, seeds, and leaves, flavonoids also have key roles in signaling between plants and microbes, in male fertility of some species, in defense as antimicrobial agents and feeding deterrents, and in UV protection. Flavonoids also have significant activities when ingested by animals, and there is great interest in their potential health benefits, particularly for compounds such as isoflavonoids, which have been linked to anticancer benefits, and stilbenes that are believed to contribute to reduced heart disease.
The major branch pathways of flavonoid biosynthesis start with general phenylpropanoid metabolism and lead to the nine major subgroups: the colorless chalcones, aurones, isoflavonoids, flavones, flavonols, flavandiols, anthocyanins, condensed tannins, and phlobaphene pigments. The enzyme phenylalanine ammonia-lyase (PAL) of the general phenylpropanoid pathway will lead to the production of cinnamic acid. Cinnamate-4-hydroxylase (C4H) will produce p-coumaric acid which will be converted through the action of 4-coumaroyl:CoA-ligase (4CL) to the production of 4-coumaroyl-CoA and malonyl-CoA. The first committed step in flavonoid biosynthesis is catalyzed by chalcone synthase (CHS), which uses malonyl CoA and 4-coumaryl CoA as substrates. Chalcone reductase (CHR) balances the production of 5-hydroxy- or 5-deoxyflavonoids. The next enzyme, chalcone isomerase (CHI) catalyses ring closure to form a flavanone, but the reaction can also occur spontaneously. Other enzymes in the pathway are: flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), flavonoid 3′-hydroxylase (F3′H) and flavonoid 3′, 5′ hydroxylase (F3′5′H).
The Arabidopsis BANYULS gene encodes a DFR-like protein that may be a leucoanthocyanidin reductase (LCR) that catalyzes an early step in condensed tannin biosynthesis. Condensed tannins are plant polyphenols with protein-precipitating and antioxidant properties, synthesized by the flavonoid pathway. Their chemical properties include protein binding, metal chelation, anti-oxidation, and UV-light absorption. As a result condensed tannins inhibit viruses, micro-organisms, insects, fungal pathogens, and monogastric digestion. Moderate amounts of tannins improve forage quality by disrupting protein foam and conferring protection from rumen pasture bloat. Bloat is a digestive disorder that occurs on some highly nutritious forage legumes such as alfalfa (Medicago sativa) and white clover (Trifolium repens). Moderate amounts of tannin can also reduce digestion rates in the rumen and can reduce parasitic load sufficiently to increase the titre of amino acids and small peptides in the small intestine without compromising total digestion.
Vestitone reductase (VR) is the penultimate enzyme in medicarpin biosynthesis. Medicarpin, a phytoalexin, has been associated with plant resistance to fungal pathogens.
While nucleic acid sequences encoding some flavonoid biosynthetic enzymes CHI, CHS, CHR, DFR, LCR, F3′5′H, F3H, F3′H, PAL and VR have been isolated for certain species of plants, there remains a need for materials useful in modifying flavonoid biosynthesis; in modifying protein binding, metal chelation, anti-oxidation, and UV-light absorption; in modifying plant pigment production; in modifying plant defense to biotic stresses such as viruses, micro-organisms, insects or fungal pathogens; in modifying forage quality, for example by disrupting protein foam and/or conferring protection from rumen pasture bloat, particularly in forage legumes and grasses, including alfalfa, medics, clovers, ryegrasses and fescues, and for methods for their use.
It is an object of the present invention to overcome, or at least alleviate, one or more of the difficulties or deficiencies associated with the prior art.
SUMMARY OF THE INVENTION
In one aspect of the present invention, substantially purified or isolated nucleic acids or nucleic acid fragments encoding amino acid sequences for a class of proteins which are related to CHI, CHS, CHR, DFR, LCR, F3′5′H, F3H, F3′H, PAL and VR and functionally active fragments and variants thereof. Such proteins are referred to herein as CHI-like, CHS-like, CHR-like, DFR-like, LCR-like, F3′5′H-like, F3H-like, F3′H-like, PAL-like and VR-like, respectively.
The individual or simultaneous enhancement or otherwise manipulation of CHI, CHS, CHR, DFR, LCR, F3′5′H, F3H, F3′H, PAL and/or VR or like gene activities in plants may enhance or otherwise alter flavonoid biosynthesis; may enhance or otherwise alter the plant capacity for protein binding, metal chelation, anti-oxidation or UV-light absorption; may enhance or reduce or otherwise alter plant pigment production; may modify plant defense to biotic stresses such as viruses, micro-organisms, insects or fungal pathogens; and/or may modify forage quality, for example by disrupting protein foam and/or conferring protection from rumen pasture bloat.
In one aspect of the present invention, substantially purified or isolated nucleic acids or fragments thereof, encoding the flavonoids biosynthetic enzymes CHI, CHS, CHR, DFR, LCR, F3′5′H, F3H, F3′H, PAL and VR from a clover (Trifolium), medic (Medicago), ryegrass (Lolium) or fescue (Festuca) species and functionally active fragments and variants thereof.
The individual or simultaneous enhancement or otherwise manipulation of CHI, CHS, CHR, DFR, LCR, F3′5′H, F3H, F3′H, PAL and/or VR or like gene activities in plants has significant consequences for a range of applications in, for example, plant production and plant protection. For example, it has applications in increasing plant tolerance and plant defense to biotic stresses such as viruses, micro-organisms, insects and fungal pathogens; in improving plant forage quality, for example by disrupting protein foam and in conferring protection from rumen pasture bloat; in reducing digestion rates in the rumen and reducing parasitic load; in the production of plant compounds leading to health benefits, such as isoflavonoids, which have been linked to anticancer benefits, and stilbenes that are believed to contribute to reduced heart disease.
Methods for the manipulation of CHI, CHS, CHR, DFR, LCR, F3′5′H, F3H, F3′H, PAL and/or VR or like gene activities in plants, including legumes such as clovers (Trifolium species), lucerne (Medicago sativa) and grass species such as ryegrasses (Lolium species) and fescues (Festuca species) may facilitate the production of, for example, forage legumes and forage grasses and other crops with enhanced tolerance to biotic stresses such as viruses, micro-organisms, insects and fungal pathogens; altered pigmentation in flowers; forage legumes with enhanced herbage quality and bloat-safety; crops with enhanced isoflavonoid content leading to health benefits.
The clover (Trifolium), medic (Medicago), ryegrass (Lolium) or fescue (Festuca) species may be of any suitable type, including white clover (Trifolium repens), red clover (Trifolium pratense), subterranean clover (Trifolium subterraneum), alfalfa (Medicago sativa), Italian or annual ryegrass (Lolium multiflorum), perennial ryegrass (Lolium perenne), tall fescue (Festuca arundinacea), meadow fescue (Festuca pratensis) and red fescue (Festuca rubra). Preferably the species is a clover or a ryegrass, more preferably white clover (T. repens) or perennial ryegrass (L. perenne). White clover (Trifolium repens L.) and perennial ryegrass (Lolium perenne L.) are key pasture legumes and grasses, respectively, in temperate climates throughout the world. Perennial ryegrass is also an important turf grass.
The nucleic acid or nucleic acid fragment may be of any suitable type and includes DNA (such as cDNA or genomic DNA) and RNA (such as mRNA) that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases, and combinations thereof.
The term “isolated” means that the material is removed from its original environment (eg. the natural environment if it is naturally occurring). For example, a naturally occurring nucleic acid present in a living plant is not isolated, but the same nucleic acid separated from some or all of the coexisting materials in the natural system, is isolated. Such nucleic acids could be part of a vector and/or such nucleic acids could be part of a composition, and still be isolated in that such a vector or composition is not part of its natural environment.
Such nucleic acids or nucleic acid fragments could be assembled to form a consensus contig. As used herein, the term “consensus contig” refers to a nucleotide sequence that is assembled from two or more constituent nucleotide sequences that share common or overlapping regions of sequence homology. For example, the nucleotide sequence of two or more nucleic acids or nucleic acid fragments can be compared and aligned in order to identify common or overlapping sequences. Where common or overlapping sequences exist between two or more nucleic acids or nucleic acid fragments, the sequences (and thus their corresponding nucleic acids or nucleic acid fragments) can be assembled into a single contiguous nucleotide sequence.
In a preferred embodiment of this aspect of the invention, the substantially purified or isolated nucleic acid or nucleic acid fragment encoding a CHI or CHI-like protein includes a nucleotide sequence selected from the group consisting of (a) sequences shown in FIGS. 1, 3, 4, 6, 7, 9, 10, 12, 122 and 127 hereto (SEQ ID NOs: 1, 3 to 7, 8, 10 to 12, 13, 15 and 16, 17, 19 to 22, 307, and 309, respectively); (b) complements of the sequences recited in (a); (c) sequences antisense to the sequences recited in (a) and (b); and (d) functionally active fragments and variants of the sequences recited in (a), (b) and (c).
In a further preferred embodiment of this aspect of the invention, the substantially purified or isolated nucleic acid or nucleic acid fragment encoding a CHS or CHS-like protein includes a nucleotide sequence selected from the group consisting of (a) sequences shown in FIGS. 13, 15, 16, 18, 19, 21, 22, 24, 25, 27, 28, 30, 31, 33, 34, 137, 142, 147, 152, 157 and 162 hereto (SEQ ID NOs: 23, 25 to 63, 64, 66 to 68, 69, 71 to 77, 78, 80 to 90, 91, 93 and 94, 95, 97 to 100, 101, 103 to 105, 106, 313, 315, 317, 319, 321, and 323, respectively); (b) complements of the sequences recited in (a); (c) sequences antisense to the sequences recited in (a) and (b); and (d) functionally active fragments and variants of the sequences recited in (a), (b) and (c).
In a further preferred embodiment of this aspect of the invention, the substantially purified or isolated nucleic acid or nucleic acid fragment encoding a CHR or CHR-like protein includes a nucleotide sequence selected from the group consisting of (a) sequences shown in FIGS. 36, 38, 40, 41, 43 and 132 hereto (SEQ ID NOs: 108, 110, 112 to 116, 117, 119 to 134, and 311, respectively); (b) complements of the sequences recited in (a); (c) sequences antisense to the sequences recited in (a) and (b); and (d) functionally active fragments and variants of the sequences recited in (a), (b) and (c).
In a further preferred embodiment of this aspect of the invention, the substantially purified or isolated nucleic acid or nucleic acid fragment encoding a DFR or DFR-like protein includes a nucleotide sequence selected from the group consisting of (a) sequences shown in FIGS. 44, 46, 47, 49, 50, 52, 54, 55, 57, 59, 61, 62, 64, 101, 103, 104, 106, 117 and 167 hereto (SEQ ID NOs: 135, 137 to 146, 147, 149 to 152, 153, 155, 157 and 158, 159, 161, 163, 165 to 167, 168, 170 to 184, 286, 288 to 292, 293, 295 to 297, 305, and 325, respectively); (b) complements of the sequences recited in (a); (c) sequences antisense to the sequences recited in (a) and (b); and (d) functionally active fragments and variants of the sequences recited in (a), (b) and (c).
In a further preferred embodiment of this aspect of the invention, the substantially purified or isolated nucleic acid or nucleic acid fragment encoding an LCR or LCR-like protein includes a nucleotide sequence selected from the group consisting of (a) sequences shown in FIGS. 65 and 67 hereto (SEQ ID NOs: 185 and 187 to 193, respectively); (b) complements of the sequences recited in (a); (c) sequences antisense to the sequences recited in (a) and (b); and (d) functionally active fragments and variants of the sequences recited in (a), (b) and (c).
In a still further preferred embodiment of this aspect of the invention, the substantially purified or isolated nucleic acid or nucleic acid fragment encoding an F3′5′H or F3′5′H-like protein includes a nucleotide sequence selected from the group consisting of (a) sequences shown in FIGS. 68, 70 and 72 hereto (SEQ ID NOs: 194, 196, and 198 to 201, respectively); (b) complements of the sequences recited in (a); (c) sequences antisense to the sequences recited in (a) and (b); and (d) functionally active fragments and variants of the sequences recited in (a), (b) and (c).
In a further preferred embodiment of this aspect of the invention, the substantially purified or isolated nucleic acid or nucleic acid fragment encoding an F3H or F3H-like protein includes a nucleotide sequence selected from the group consisting of (a) sequences shown in FIGS. 73, 75, 76, 78, 107, 109, 111 and 172 hereto (SEQ ID NOs: 202, 204 to 244, 245, 247, 298, 300 to 302, 303, and 327, respectively); (b) complements of the sequences recited in (a); (c) sequences antisense to the sequences recited in (a) and (b); and (d) functionally active fragments and variants of the sequences recited in (a), (b) and (c).
In a still further preferred embodiment of this aspect of the invention, the substantially purified or isolated nucleic acid or nucleic acid fragment encoding an F3′H or F3′H-like protein includes a nucleotide sequence selected from the group consisting of (a) sequences shown in FIGS. 80 and 82 hereto (SEQ ID NOs: 249, and 251 and 252, respectively); (b) complements of the sequences recited in (a); (c) sequences antisense to the sequences recited in (a) and (b); and (d) functionally active fragments and variants of the sequences recited in (a), (b) and (c).
In a further preferred embodiment of this aspect of the invention, the substantially purified or isolated nucleic acid or nucleic acid fragment encoding an PAL or PAL-like protein includes a nucleotide sequence selected from the group consisting of (a) sequences shown in FIGS. 83, 85, 86, 88, 89, 91, 93, 95, 97, 177, 182 and 187 hereto (SEQ ID NOs: 253, 255 to 257, 258, 260 to 267, 268, 270, 272, 274, 276 and 277, 329, 331, and 333, respectively); (b) complements of the sequences recited in (a); (c) sequences antisense to the sequences recited in (a) and (b); and (d) functionally active fragments and variants of the sequences recited in (a), (b) and (c).
In a still further preferred embodiment of this aspect of the invention, the substantially purified or isolated nucleic acid or nucleic acid fragment encoding an VR or VR-like protein includes a nucleotide sequence selected from the group consisting of (a) sequences shown in FIGS. 98, 100 and 192 hereto (SEQ ID NOs: 278, 280 to 285, and 335, respectively); (b) complements of the sequences recited in (a); (c) sequences antisense to the sequences recited in (a) and (b); and (d) functionally active fragments and variants of the sequences recited in (a), (b) and (c).
By “functionally active” in relation to nucleic acids it is meant that the fragment or variant (such as an analogue, derivative or mutant) encodes a polypeptide which is capable of modifying flavonoid biosynthesis in a plant. Such variants include naturally occurring allelic variants and non-naturally occurring variants. Additions, deletions, substitutions and derivatizations of one or more of the nucleotides are contemplated so long as the modifications do not result in loss of functional activity of the fragment or variant. Preferably the functionally active fragment or variant has at least approximately 80% identity to the relevant part of the above mentioned nucleotide sequence, more preferably at least approximately 90% identity, even more preferably at least approximately 95% identity, most preferably at least approximately 98% homology. Such functionally active variants and fragments include, for example, those having nucleic acid changes which result in conservative amino acid substitutions of one or more residues in the corresponding amino acid sequence. Preferably the fragment has a size of at least 10 nucleotides, more preferably at least 15 nucleotides, most preferably at least 20 nucleotides.
Nucleic acids or nucleic acid fragments encoding at least a portion of several CHI, CHS, CHR, DFR, LCR, F3′5′H, F3H, F3′H, PAL and VR have been isolated and identified. The nucleic acids or nucleic acid fragments of the present invention may be used to isolate cDNAs and genes encoding homologous proteins from the same or other plant species. Isolation of homologous genes using sequence-dependent protocols, such as methods of nucleic acid hybridisation, and methods of DNA and RNA amplification as exemplified by various uses of nucleic acid amplification technologies (e.g. polymerase chain reaction, ligase chain reaction), is well known in the art.
For example, genes encoding other CHI or CHI-like, CHS or CHS-like, CHR or CHR-like, DFR or DFR-like, LCR or LCR-like; F3′5′H or F3′5′H-like, F3H or F3H-like, F3′H or F3′H-like, PAL or PAL-like and VR or VR-like proteins, either as cDNAs or genomic DNAs, may be isolated directly by using all or a portion of the nucleic acids or nucleic acid fragments of the present invention as hybridisation probes to screen libraries from the desired plant employing the methodology well known to those skilled in the art. Specific oligonucleotide probes based upon the nucleic acid sequences of the present invention may be designed and synthesized by methods known in the art. Moreover, the entire sequences may be used directly to synthesize DNA probes by methods known to the skilled artisan such as random primer DNA labelling, nick translation, or end-labelling techniques, or RNA probes using available in vitro transcription systems. In addition, specific primers may be designed and used to amplify a part or all of the sequences of the present invention. The resulting amplification products may be labelled directly during amplification reactions or labelled after amplification reactions, and used as probes to isolate full-length cDNA or genomic fragments under conditions of appropriate stringency.
In addition, short segments of the nucleic acids or nucleic acid fragments of the present invention may be used in amplification protocols to amplify longer nucleic acids or nucleic acid fragments encoding homologous genes from DNA or RNA. For example, polymerase chain reaction may be performed on a library of cloned nucleic acid fragments wherein the sequence of one primer is derived from the nucleic acid sequences of the present invention, and the sequence of the other primer takes advantage of the presence of the polyadenylic acid tracts to the 3′ end of the mRNA precursor encoding plant genes. Alternatively, the second primer sequence may be based upon sequences derived from the cloning vector. For example, those skilled in the art can follow the RACE protocol [Frohman et al. (1988) Proc. Natl. Acad Sci. USA 85:8998, the entire disclosure of which is incorporated herein by reference] to generate cDNAs by using PCR to amplify copies of the region between a single point in the transcript and the 3′ or 5′ end. Using commercially available 3′ RACE and 5′ RACE systems (BRL), specific 3′ or 5′ cDNA fragments may be isolated [Ohara et al. (1989) Proc. Natl. Acad Sci USA 86:5673; Loh et al. (1989) Science 243:217, the entire disclosures of which are incorporated herein by reference]. Products generated by the 3′ and 5′ RACE procedures may be combined to generate full-length cDNAs.
In a second aspect of the present invention there is provided a substantially purified or isolated polypeptide from a clover (Trifolium), medic (Medicago), ryegrass (Lolium) or fescue (Festuca) species, selected from the group consisting of CHI and CHI-like, CHS and CHS-like, CHR and CHR-like, DFR and DFR-like, LCR and LCR-like, F3′5′H and F3′5′H-like, F3H and F3H-like, F3′H and F3′H-like, PAL and PAL-like, VR and VR-like; and functionally active fragments and variants thereof.
The clover (Trifolium), medic (Medicago), ryegrass (Lolium) or fescue (Festuca) species may be of any suitable type, including white clover (Trifolium repens), red clover (Trifolium pratense), subterranean clover (Trifolium subterraneum), alfalfa (Medicago sativa), Italian or annual ryegrass (Lolium multiflorum), perennial ryegrass (Lolium perenne), tall fescue (Festuca arundinacea), meadow fescue (Festuca pratensis) and red fescue (Festuca rubra). Preferably the species is a clover or a ryegrass, more preferably white clover (T. repens) or perennial ryegrass (L. perenne).
In a preferred embodiment of this aspect of the invention, the substantially purified or isolated CHI or CHI-like polypeptide includes an amino acid sequence selected from the group consisting of the sequences shown in FIGS. 2, 5, 8, 11, 123 and 128 hereto (SEQ ID NOs: 2, 9, 14, 18, 308, and 310, respectively), and functionally active fragments and variants thereof.
In a further preferred embodiment of this aspect of the invention, the substantially purified or isolated CHS or CHS-like polypeptide includes an amino acid sequence selected from the group consisting of the sequences shown in FIGS. 14, 17, 20, 23, 26, 29, 32, 35, 138, 143, 148, 153, 158 and 163 hereto (SEQ ID NOs: 24, 65, 70, 79, 92, 96, 102, 107, 314, 316, 318, 320, 322, and 324, respectively), and functionally active fragments and variants thereof.
In a further preferred embodiment of this aspect of the invention, the substantially purified or isolated CHR or CHR-like polypeptide includes an amino acid sequence selected from the group consisting of the sequences shown in FIGS. 37, 39, 42 and 133 hereto (SEQ ID NOs: 109, 111, 118, and 312, respectively), and functionally active fragments and variants thereof.
In a still further preferred embodiment of this aspect of the invention, the substantially purified or isolated DFR or DFR-like polypeptide includes an amino acid sequence selected from the group consisting of the sequences shown in FIGS. 45, 48, 51, 53, 56, 58, 60, 63, 102, 105, 118 and 168 hereto (SEQ ID NOs: 136, 148, 54, 156, 160, 162, 164, 169, 287, 294, 306, and 326, respectively), and functionally active fragments and variants thereof.
In a still further preferred embodiment of this aspect of the invention, the substantially purified or isolated LCR or LCR-like polypeptide includes an amino acid sequence shown in FIG. 66 hereto (SEQ ID NO: 186), and functionally active fragments and variants thereof.
In a still further preferred embodiment of this aspect of the invention, the substantially purified or isolated F3′5′H or F3′5′H-like polypeptide includes an amino acid sequence selected from the group consisting of the sequences shown in FIGS. 69 and 71 hereto (SEQ ID NOs: 195 and 197, respectively), and functionally active fragments and variants thereof.
In a still further preferred embodiment of this aspect of the invention, the substantially purified or isolated F3H or F3H-like polypeptide includes an amino acid sequence selected from the group consisting of the sequences shown in FIGS. 74, 77, 79, 108, 112 and 173 hereto (SEQ ID NOs: 203, 246, 248, 299, 304, and 328, respectively), and functionally active fragments and variants thereof.
In a still further preferred embodiment of this aspect of the invention, the substantially purified or isolated F3′H or F3′H-like polypeptide includes an amino acid sequence shown in FIG. 81 hereto (SEQ ID NO: 250), and functionally active fragments and variants thereof.
In a still further preferred embodiment of this aspect of the invention, the substantially purified or isolated PAL or PAL-like polypeptide includes an amino acid sequence selected from the group consisting of the sequences shown in FIGS. 84, 87, 90, 92, 94, 96, 178, 183 and 188 hereto (SEQ ID NOs: 254, 259, 269, 271, 273, 275, 330, 332, and 334, respectively), and functionally active fragments and variants thereof.
In a still further preferred embodiment of this aspect of the invention, the substantially purified or isolated VR or VR-like polypeptide includes an amino acid sequence shown in FIGS. 99 and 193 hereto (SEQ ID NOs: 279 and 336, respectively), and functionally active fragments and variants thereof.
By “functionally active” in relation to polypeptides it is meant that the fragment or variant has one or more of the biological properties of the proteins CHI, CHI-like, CHS, CHS-like, CHR, CHR-like, DFR, DFR-like, LCR, LCR-like, F3′5′H, F3′5′H-like, F3H, F3H-like, F3′H, F3′H-like, PAL, PAL-like, VR and VR-like, respectively. Additions, deletions, substitutions and derivatizations of one or more of the amino acids are contemplated so long as the modifications do not result in loss of functional activity of the fragment or variant. Preferably the functionally active fragment or variant has at least approximately 60% identity to the relevant part of the above mentioned amino acid sequence, more preferably at least approximately 80% identity, even more preferably at least approximately 90% identity most preferably at least approximately 95% homology. Such functionally active variants and fragments include, for example, those having conservative amino acid substitutions of one or more residues in the corresponding amino acid sequence. Preferably the fragment has a size of at least 10 amino acids, more preferably at least 15 amino acids, most preferably at least 20 amino acids.
In a further embodiment of this aspect of the invention, there is provided a polypeptide recombinantly produced from a nucleic acid or nucleic acid fragment according to the present invention. Techniques for recombinantly producing polypeptides are well known to those skilled in the art.
Availability of the nucleotide sequences of the present invention and deduced amino acid sequences facilitates immunological screening of cDNA expression libraries. Synthetic peptides representing portions of the instant amino acid sequences may be synthesized. These peptides may be used to immunise animals to produce polyclonal or monoclonal antibodies with specificity for peptides and/or proteins including the amino acid sequences. These antibodies may be then used to screen cDNA expression libraries to isolate full-length cDNA clones of interest.
A genotype is the genetic constitution of an individual or group. Variations in genotype are important in commercial breeding programs, in determining parentage, in diagnostics and fingerprinting, and the like. Genotypes can be readily described in terms of genetic markers. A genetic marker identifies a specific region or locus in the genome. The more genetic markers, the finer defined is the genotype. A genetic marker becomes particularly useful when it is allelic between organisms because it then may serve to unambiguously identify an individual. Furthermore, a genetic marker becomes particularly useful when it is based on nucleic acid sequence information that can unambiguously establish a genotype of an individual and when the function encoded by such nucleic acid is known and is associated with a specific trait. Such nucleic acids and/or nucleotide sequence information including single nucleotide polymorphisms (SNPs), variations in single nucleotides between allelic forms of such nucleotide sequence, may be used as perfect markers or candidate genes for the given trait.
Applicants have identified a number of SNPs of the nucleic acids or nucleic acid fragments of the present invention. These are indicated (marked with grey on the black background) in the figures that show multiple alignments of nucleotide sequences of nucleic acid fragments contributing to consensus contig sequences. See for example, FIGS. 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 40, 43, 46, 49, 54, 61, 64, 67, 72, 75, 82, 85, 88, 97, 100, 103, 106 and 109 hereto (SEQ ID NOs: 3 to 7, 10 to 12, 15 and 16, 19 to 22, 25 to 63, 66 to 68, 71 to 77, 80 to 90, 93 and 94, 97 to 100, 103 to 105, 112 to 116, 119 to 134, 137 to 146, 149 to 152, 157 and 158, 165 to 167, 170 to 184, 187 to 193, 198 to 201, 204 to 244, 251 and 252, 255 to 257, 260 to 267, 276 and 277, 280 to 285, 288 to 292, 295 to 297, and 300 to 302, respectively).
Accordingly, in a further aspect of the present invention, there is provided a substantially purified or isolated nucleic acid or nucleic acid fragment including a single nucleotide polymorphism (SNP) from a nucleic acid or nucleic acid fragment according to the present invention or complements or sequences antisense thereto, and functionally active fragments and variants thereof.
In a still further aspect of the present invention there is provided a method of isolating a nucleic acid or nucleic acid fragment of the present invention including a SNP, said method including sequencing nucleic acid fragments from a nucleic acid library.
The nucleic acid library may be of any suitable type and is preferably a cDNA library.
The nucleic acid or nucleic acid fragments may be isolated from a recombinant plasmid or may be amplified, for example using polymerase chain reaction.
The sequencing may be performed by techniques known to those skilled in the art.
In a still further aspect of the present invention, there is provided use of the nucleic acids or nucleic acid fragments of the present invention including SNPs, and/or nucleotide sequence information thereof, as molecular genetic markers.
In a still further aspect of the present invention there is provided use of a nucleic acid or nucleic acid fragment of the present invention, and/or nucleotide sequence information thereof, as a molecular genetic marker.
More particularly, nucleic acids or nucleic acid fragments according to the present invention and/or nucleotide sequence information thereof may be used as a molecular genetic marker for quantitative trait loci (QTL) tagging, QTL mapping, DNA fingerprinting and in marker assisted selection, particularly in clovers, alfalfa, ryegrasses and fescues. Even more particularly, nucleic acids or nucleic acid fragments according to the present invention and/or nucleotide sequence information thereof may be used as molecular genetic markers in plant improvement in relation to plant tolerance to biotic stresses such as viruses, micro-organisms, insects, fungal pathogens; in relation to forage quality; in relation to bloat safety; in relation to condensed tannin content; in relation to plant pigmentation. Even more particularly, sequence information revealing SNPs in allelic variants of the nucleic acids or nucleic acid fragments of the present invention and/or nucleotide sequence information thereof may be used as molecular genetic markers for QTL tagging and mapping and in marker assisted selection, particularly in clovers, alfalfa, ryegrasses and fescues.
In a still further aspect of the present invention there is provided a construct including a nucleic acid or nucleic acid fragment according to the present invention.
The term “construct” as used herein refers to an artificially assembled or isolated nucleic acid molecule which includes the gene of interest. In general a construct may include the gene or genes of interest, a marker gene which in some cases can also be the gene of interest and appropriate regulatory sequences. It should be appreciated that the inclusion of regulatory sequences in a construct is optional, for example, such sequences may not be required in situations where the regulatory sequences of a host cell are to be used. The term construct includes vectors but should not be seen as being limited thereto.
In a still further aspect of the present invention there is provided a vector including a nucleic acid or nucleic acid fragment according to the present invention.
The term “vector” as used herein encompasses both cloning and expression vectors. Vectors are often recombinant molecules containing nucleic acid molecules from several sources.
In a preferred embodiment of this aspect of the invention, the vector may include a regulatory element such as a promoter, a nucleic acid or nucleic acid fragment according to the present invention and a terminator; said regulatory element, nucleic acid or nucleic acid fragment and terminator being operatively linked.
By “operatively linked” is meant that said regulatory element is capable of causing expression of said nucleic acid or nucleic acid fragment in a plant cell and said terminator is capable of terminating expression of said nucleic acid or nucleic acid fragment in a plant cell. Preferably, said regulatory element is upstream of said nucleic acid or nucleic acid fragment and said terminator is downstream of said nucleic acid or nucleic acid fragment.
The vector may be of any suitable type and may be viral or non-viral. The vector may be an expression vector. Such vectors include chromosomal, non-chromosomal and synthetic nucleic acid sequences, e.g. derivatives of plant viruses; bacterial plasmids; derivatives of the Ti plasmid from Agrobacterium tumefaciens, derivatives of the Ri plasmid from Agrobacterium rhizogenes; phage DNA; yeast artificial chromosomes; bacterial artificial chromosomes; binary bacterial artificial chromosomes; vectors derived from combinations of plasmids and phage DNA. However, any other vector may be used as long as it is replicable, integrative or viable in the plant cell.
The regulatory element and terminator may be of any suitable type and may be endogenous to the target plant cell or may be exogenous, provided that they are functional in the target plant cell.
Preferably the regulatory element is a promoter. A variety of promoters which may be employed in the vectors of the present invention are well known to those skilled in the art. Factors influencing the choice of promoter include the desired tissue specificity of the vector, and whether constitutive or inducible expression is desired and the nature of the plant cell to be transformed (e.g. monocotyledon or dicotyledon). Particularly suitable constitutive promoters include the Cauliflower Mosaic Virus 35S (CaMV 35S) promoter and derivatives thereof, the maize Ubiquitin promoter, and the rice Actin promoter.
A variety of terminators which may be employed in the vectors of the present invention are also well known to those skilled in the art. The terminator may be from the same gene as the promoter sequence or a different gene. Particularly suitable terminators are polyadenylation signals, such as the CaMV 35S polyA and other terminators from the nopaline synthase (nos), the octopine synthase (ocs) and the rbcS genes.
The vector, in addition to the regulatory element, the nucleic acid or nucleic acid fragment of the present invention and the terminator, may include further elements necessary for expression of the nucleic acid or nucleic acid fragment, in different combinations, for example vector backbone, origin of replication (ori), multiple cloning sites, spacer sequences, enhancers, introns (such as the maize Ubiquitin Ubi intron), antibiotic resistance genes and other selectable marker genes [such as the neomycin phosphotransferase (npt2) gene, the hygromycin phosphotransferase (hph) gene, the phosphinothricin acetyltransferase (bar or pat) gene and the gentamycin acetyl transferase (aacC1) gene], and reporter genes [such as beta-glucuronidase (GUS) gene (gusA) and green fluorescent protein (gfp)]. The vector may also contain a ribosome binding site for translation initiation. The vector may also include appropriate sequences for amplifying expression.
As an alternative to use of a selectable marker gene to provide a phenotypic trait for selection of transformed host cells, the presence of the vector in transformed cells may be determined by other techniques well known in the art, such as PCR (polymerase chain reaction), Southern blot hybridisation analysis, histochemical GUS assays, northern and Western blot hybridisation analyses.
Those skilled in the art will appreciate that the various components of the vector are operatively linked, so as to result in expression of said nucleic acid or nucleic acid fragment. Techniques for operatively linking the components of the vector of the present invention are well known to those skilled in the art. Such techniques include the use of linkers, such as synthetic linkers, for example including one or more restriction enzyme sites.
The constructs and vectors of the present invention may be incorporated into a variety of plants, including monocotyledons (such as grasses from the genera Lolium, Festuca, Paspalum, Pennisetum, Panicum and other forage and turfgrasses, corn, oat, sugarcane, wheat and barley), dicotyledons (such as Arabidopsis, tobacco, clovers, medics, eucalyptus, potato, sugarbeet, canola, soybean, chickpea) and gymnosperms. In a preferred embodiment, the constructs and vectors may be used to transform monocotyledons, preferably grass species such as ryegrasses (Lolium species) and fescues (Festuca species), more preferably perennial ryegrass, including forage- and turf-type cultivars. In an alternate preferred embodiment, the constructs and vectors may be used to transform dicotyledons, preferably forage legume species such as clovers (Trifolium species) and medics (Medicago species), more preferably white clover (Trifolium repens), red clover (Trifolium pratense), subterranean clover (Trifolium subterraneum) and alfalfa (Medicago sativa). Clovers, alfalfa and medics are key pasture legumes in temperate climates throughout the world.
Techniques for incorporating the constructs and vectors of the present invention into plant cells (for example by transduction, transfection or transformation) are well known to those skilled in the art. Such techniques include Agrobacterium mediated introduction, electroporation to tissues, cells and protoplasts, protoplast fusion, injection into reproductive organs, injection into immature embryos and high velocity projectile introduction to cells, tissues, calli, immature and mature embryos. The choice of technique will depend largely on the type of plant to be transformed.
Cells incorporating the constructs and vectors of the present invention may be selected, as described above, and then cultured in an appropriate medium to regenerate transformed plants, using techniques well known in the art. The culture conditions, such as temperature, pH and the like, will be apparent to the person skilled in the art. The resulting plants may be reproduced, either sexually or asexually, using methods well known in the art, to produce successive generations of transformed plants.
In a further aspect of the present invention there is provided a plant cell, plant, plant seed or other plant part, including, e.g. transformed with, a construct, vector, nucleic acid or nucleic acid fragment of the present invention.
The plant cell, plant, plant seed or other plant part may be from any suitable species, including monocotyledons, dicotyledons and gymnosperms. In a preferred embodiment the plant cell, plant, plant seed or other plant part may be from a monocotyledon, preferably a grass species, more preferably a ryegrass (Lolium species) or fescue (Festuca species), even more preferably perennial ryegrass, including both forage- and turf-type cultivars. In an alternate preferred embodiment the plant cell, plant, plant seed or other plant part may be from a dicotyledon, preferably forage legume species such as clovers (Trifolium species) and medics (Medicago species), more preferably white clover (Trifolium repens), red clover (Trifolium pratense), subterranean clover (Trifolium subterraneum) and alfalfa (Medicago sativa).
The present invention also provides a plant, plant seed or other plant part, or a plant extract derived from a plant cell of the present invention.
The present invention also provides a plant, plant seed or other plant part, or a plant extract derived from a plant of the present invention.
In a further aspect of the present invention there is provided a method of modifying flavonoid biosynthesis in a plant; said method including introducing into said plant an effective amount of a nucleic acid or nucleic acid fragment, construct and/or a vector according to the present invention.
In a further aspect of the present invention there is provided a method of modifying protein binding, metal chelation, anti-oxidation, and/or UV-light absorption in a plant, said method including introducing into said plant an effective amount of a nucleic acid or nucleic acid fragment, construct and/or a vector according to the present invention.
In a further aspect of the present invention there is provided a method of modifying pigment production in a plant, said method including introducing into said plant an effective amount of a nucleic acid or nucleic acid fragment, construct and/or a vector according to the present invention.
In a further aspect of the present invention there is provided a method of modifying plant defense to biotic stresses such as viruses, micro-organisms, insects and fungal pathogens, said method including introducing into said plant an effective amount of a nucleic acid or nucleic acid fragment, construct and/or a vector according to the present invention.
In a further aspect of the present invention there is provided a method of modifying forage quality of a plant by disrupting protein foam and/or conferring protection from rumen pasture bloat, said method including introducing into said plant an effective amount of a nucleic acid or nucleic acid fragment, construct and/or a vector according to the present invention.
By “an effective amount” it is meant an amount sufficient to result in an identifiable phenotypic trait in said plant, or a plant, plant seed or other plant part derived therefrom. Such amounts can be readily determined by an appropriately skilled person, taking into account the type of plant, the route of administration and other relevant factors. Such a person will readily be able to determine a suitable amount and method of administration. See, for example, Maniatis et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, the entire disclosure of which is incorporated herein by reference.
Using the methods and materials of the present invention, flavonoid biosynthesis, protein binding, metal chelation, anti-oxidation, UV-light absorption, tolerance to biotic stresses such as viruses, micro-organisms, insects and fungal pathogens; pigmentation in for example flowers and leaves; herbage quality and bloat-safety; and/or isoflavonoid content leading to health benefits, may be increased or otherwise modified, for example by incorporating additional copies of a sense nucleic acid or nucleic acid fragment of the present invention. They may be decreased or otherwise modified, for example by incorporating an antisense nucleic acid or nucleic acid fragment of the present invention.
The present invention will now be more fully described with reference to the accompanying Examples and drawings. It should be understood, however, that the description following is illustrative only and should not be taken in any way as a restriction on the generality of the invention described above.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the consensus contig nucleotide sequence of TrCHIa (SEQ ID NO: 1).
FIG. 2 shows the deduced amino acid sequence of TrCHIa (SEQ ID NO: 2).
FIG. 3A and FIG. 3B show the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence TrCHIa (TrCHIa1: SEQ ID NO: 3; TrCHIa2: SEQ ID NO: 4; TrCHIa3: SEQ ID NO: 5; TrCHIa4: SEQ ID NO: 6; TrCHIa5: SEQ ID NO: 7).
FIG. 4 shows the consensus contig nucleotide sequence of TrCHIb (SEQ ID NO: 8).
FIG. 5 shows the deduced amino acid sequence of TrCHIb (SEQ ID NO: 9).
FIG. 6 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence TrCHIb (TrCHIb1: SEQ ID NO: 10, TrCHIb2: SEQ ID NO: 11; TrCHIb3: SEQ ID NO: 12).
FIG. 7 shows the consensus contig nucleotide sequence of TrCHIc (SEQ ID NO: 13).
FIG. 8 shows the deduced amino acid sequence of TrCHIc (SEQ ID NO: 14).
FIG. 9 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence TrCHIc (TrCHIc1: SEQ ID NO: 15; TrCHIc2: SEQ ID NO: 16).
FIG. 10 shows the consensus contig nucleotide sequence of TrCHId (SEQ ID NO: 17).
FIG. 11 shows the deduced amino acid sequence of TrCHId (SEQ ID NO: 18).
FIG. 12A and FIG. 12B show the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence TrCHId (TrCHId1: SEQ ID NO: 19; TrCHId2: SEQ ID NO: 20; TrCHId3: SEQ ID NO: 21; TrCHId4: SEQ ID NO: 22).
FIG. 13 shows the consensus contig nucleotide sequence of TrCHSa (SEQ ID NO: 23).
FIG. 14 shows the deduced amino acid sequence of TrCHSa (SEQ ID NO: 24).
FIG. 15A-FIG. 15S show the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence TrCHSa (TrCHSa1: SEQ ID NO: 25; TrCHSa2: SEQ ID NO: 26; TrCHSa3: SEQ ID NO: 27; TrCHSa4: SEQ ID NO: 28; TrCHSa5: SEQ ID NO: 29; TrCHSa6: SEQ ID NO: 30; TrCHSa7: SEQ ID NO: 31; TrCHSa8: SEQ ID NO: 32; TrCHSa9: SEQ ID NO: 33; TrCHSa10: SEQ ID NO: 34; TrCHSa11: SEQ ID NO: 35; TrCHSa12: SEQ ID NO: 36; TrCHSa13: SEQ ID NO: 37; TrCHSa14: SEQ ID NO: 38; TrCHSa15: SEQ ID NO: 39; TrCHSa16: SEQ ID NO: 40; TrCHSa17: SEQ ID NO: 41; TrCHSa18: SEQ ID NO: 42; TrCHSa19: SEQ ID NO: 43; TrCHSa20: SEQ ID NO: 44; TrCHSa21: SEQ ID NO: 45; TrCHSa22: SEQ ID NO: 46; TrCHSa23: SEQ ID NO: 47; TrCHSa24: SEQ ID NO: 48; TrCHSa25: SEQ ID NO: 49; TrCHSa26: SEQ ID NO: 50; TrCHSa27: SEQ ID NO: 51; TrCHSa28: SEQ ID NO: 52; TrCHSa29: SEQ ID NO: 53; TrCHSa30: SEQ ID NO: 54; TrCHSa31: SEQ ID NO: 55; TrCHSa32: SEQ ID NO: 56; TrCHSa33: SEQ ID NO: 57; TrCHSa34: SEQ ID NO: 58; TrCHSa35: SEQ ID NO: 59; TrCHSa36: SEQ ID NO: 60; TrCHSa37: SEQ ID NO: 61; TrCHSa38: SEQ ID NO: 62; TrCHSa39: SEQ ID NO: 63).
FIG. 16 shows the consensus contig nucleotide sequence of TrCHSb (SEQ ID NO: 64).
FIG. 17 shows the deduced amino acid sequence of TrCHSb (SEQ ID NO: 65).
FIG. 18 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence TrCHSb (TrCHSb1: SEQ ID NO: 66; TrCHSb2: SEQ ID NO: 67; TrCHSb3: SEQ ID NO: 68).
FIG. 19 shows the consensus contig nucleotide sequence of TrCHSc (SEQ ID NO: 69).
FIG. 20 shows the deduced amino acid sequence of TrCHSc (SEQ ID NO: 70).
FIG. 21A-FIG. 21C show the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence TrCHSc (TrCHSc 1: SEQ ID NO: 71; TrCHSc2: SEQ ID NO: 72; TrCHSc3: SEQ ID NO: 73; TrCHSc4: SEQ ID NO: 74; TrCHSc5: SEQ ID NO: 75; TrCHSc6: SEQ ID NO: 76; TrCHSc7: SEQ ID NO: 77).
FIG. 22 shows the consensus contig nucleotide sequence of TrCHSd (SEQ ID NO: 78).
FIG. 23 shows the deduced amino acid sequence of TrCHSd (SEQ ID NO: 79).
FIG. 24A-FIG. 24D show the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence TrCHSd (TrCHSd1: SEQ ID NO: 80; TrCHSd1: SEQ ID NO: 81; TrCHSd1: SEQ ID NO: 82; TrCHSd1: SEQ ID NO: 83; TrCHSd1: SEQ ID NO: 84; TrCHSd1: SEQ ID NO: 85; TrCHSd1: SEQ ID NO: 86; TrCHSd1: SEQ ID NO: 87; TrCHSd1: SEQ ID NO: 88; TrCHSd1: SEQ ID NO: 89; TrCHSd1: SEQ ID NO: 90).
FIG. 25 shows the consensus contig nucleotide sequence of TrCHSe (SEQ ID NO: 91).
FIG. 26 shows the deduced amino acid sequence of TrCHSe (SEQ ID NO: 92).
FIG. 27 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence TrCHSe (TrCHSe1: SEQ ID NO: 93; TrCHSe2: SEQ ID NO: 94).
FIG. 28 shows the consensus contig nucleotide sequence of TrCHSf (SEQ ID NO: 95).
FIG. 29 shows the deduced amino acid sequence of TrCHSf (SEQ ID NO: 96).
FIG. 30A and FIG. 30B show the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence TrCHSf (TrCHSf1: SEQ ID NO: 97; TrCHSf2: SEQ ID NO: 98; TrCHSf1: SEQ ID NO: 99; TrCHSf1: SEQ ID NO: 100).
FIG. 31 shows the consensus contig nucleotide sequence of TrCHSg (SEQ ID NO: 101).
FIG. 32 shows the deduced amino acid sequence of TrCHSg (SEQ ID NO: 102).
FIG. 33A and FIG. 33B show the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence TrCHSg (TrCHSg1: SEQ ID NOs: 103; TrCHSg2: SEQ ID NOs: 104; TrCHSg3: SEQ ID NOs: 105).
FIG. 34 shows the consensus contig nucleotide sequence of TrCHSh (SEQ ID NO: 106).
FIG. 35 shows the deduced amino acid sequence of TrCHSh (SEQ ID NO: 107).
FIG. 36 shows the nucleotide sequence of TrCHRa (SEQ ID NO: 108).
FIG. 37 shows the deduced amino acid sequence of TrCHRa (SEQ ID NO: 109).
FIG. 38 shows the consensus contig nucleotide sequence of TrCHRb (SEQ ID NO: 110).
FIG. 39 shows the deduced amino acid sequence of TrCHRb (SEQ ID NO: 111).
FIG. 40A and FIG. 40B show the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence TrCHRb (TrCHRb1: SEQ ID NO: 112; TrCHRb2: SEQ ID NO: 113; TrCHRb3: SEQ ID NO: 114; TrCHRb4: SEQ ID NO: 115; TrCHRb5: SEQ ID NO: 116).
FIG. 41 shows the consensus contig nucleotide sequence of TrCHRc (SEQ ID NO: 117).
FIG. 42 shows the deduced amino acid sequence of TrCHRc (SEQ ID NO: 118).
FIG. 43A-FIG. 43D show the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence TrCHRc (TrCHRc1: SEQ ID NO: 119; TrCHRc2: SEQ ID NO: 120; TrCHRc3: SEQ ID NO: 121; TrCHRc4: SEQ ID NO: 122; TrCHRc5: SEQ ID NO: 123; TrCHRc6: SEQ ID NO: 124; TrCHRc7: SEQ ID NO: 125; TrCHRc8: SEQ ID NO: 126; TrCHRc9: SEQ ID NO: 127; TrCHRc10: SEQ ID NO: 128; TrCHRc11: SEQ ID NO: 129; TrCHRc12: SEQ ID NO: 130; TrCHRc13: SEQ ID NO: 131; TrCHRc14: SEQ ID NO: 132; TrCHRc15: SEQ ID NO: 133; TrCHRc16: SEQ ID NO: 134).
FIG. 44 shows the consensus contig nucleotide sequence of TrDFRa (SEQ ID NO: 135).
FIG. 45 shows the deduced amino acid sequence of TrDFRa (SEQ ID NO: 136).
FIG. 46A-FIG. 46C show the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence TrDFRa (TrDFRa1: SEQ ID NO: 137; TrDFRa2: SEQ ID NO: 138; TrDFRa3: SEQ ID NO: 139; TrDFRa4: SEQ ID NO: 140; TrDFRa5: SEQ ID NO: 141; TrDFRa6: SEQ ID NO: 142; TrDFRa7: SEQ ID NO: 143; TrDFRa8: SEQ ID NO: 144; TrDFRa9: SEQ ID NO: 145; TrDFRa10: SEQ ID NO: 146).
FIG. 47 shows the consensus contig nucleotide sequence of TrDFRb (SEQ ID NO: 147).
FIG. 48 shows the deduced amino acid sequence of TrDFRb (SEQ ID NO: 148).
FIG. 49A and FIG. 49B show the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence TrDFRb (TrDFRb1: SEQ ID NO: 149; TrDFRb2: SEQ ID NO: 150; TrDFRb3: SEQ ID NO: 151; TrDFRb4: SEQ ID NO: 152).
FIG. 50 shows the nucleotide sequence of TrDFRc (SEQ ID NO: 153).
FIG. 51 shows the deduced amino acid sequence of TrDFRc (SEQ ID NO: 154).
FIG. 52 shows the consensus contig nucleotide sequence of TrDFRd (SEQ ID NO: 155).
FIG. 53 shows the deduced amino acid sequence of TrDFRd (SEQ ID NO: 156).
FIG. 54A and FIG. 54B show the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence TrDFRd (TrDFRd1: SEQ ID NO: 157; TrDFRd2: SEQ ID NO: 158).
FIG. 55 shows the nucleotide sequence of TrDFRe (SEQ ID NO: 159).
FIG. 56 shows the deduced amino acid sequence of TrDFRe (SEQ ID NO: 160).
FIG. 57 shows the nucleotide sequence of TrDFRf (SEQ ID NO: 161).
FIG. 58 shows the deduced amino acid sequence of TrDFRf (SEQ ID NO: 162).
FIG. 59 shows the consensus contig nucleotide sequence of TrDFRg (SEQ ID NO: 163).
FIG. 60 shows the deduced amino acid sequence of TrDFRg (SEQ ID NO: 164).
FIG. 61A and FIG. 61B show the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence TrDFRg (TrDFRg1: SEQ ID NO: 165; TrDFRg2: SEQ ID NO: 166; TrDFRg3: SEQ ID NO: 167).
FIG. 62A and FIG. 62B show the consensus contig nucleotide sequence of TrDFRh (SEQ ID NO: 168).
FIG. 63 shows the deduced amino acid sequence of TrDFRh (SEQ ID NO: 169).
FIG. 64A-FIG. 64F show the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence TrDFRh (TrDFRh1: SEQ ID NO: 170; TrDFRh2: SEQ ID NO: 171; TrDFRh3: SEQ ID NO: 172; TrDFRh4: SEQ ID NO: 173; TrDFRh5: SEQ ID NO: 174; TrDFRh6: SEQ ID NO: 175; TrDFRh7: SEQ ID NO: 176; TrDFRh8: SEQ ID NO: 177; TrDFRh9: SEQ ID NO: 178; TrDFRh10: SEQ ID NO: 179; TrDFRh11: SEQ ID NO: 180; TrDFRh12: SEQ ID NO: 181; TrDFRh13: SEQ ID NO: 182; TrDFRh14: SEQ ID NO: 183; TrDFRh 15: SEQ ID NO: 184).
FIG. 65 shows the consensus contig nucleotide sequence of TrLCRa (SEQ ID NO: 185).
FIG. 66 shows the deduced amino acid sequence of TrLCRa (SEQ ID NO: 186).
FIG. 67A-FIG. 67C show the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence TrLCRa (TrLCRa1: SEQ ID NO: 187; TrLCRa2: SEQ ID NO: 188; TrLCRa3: SEQ ID NO: 189; TrLCRa4: SEQ ID NO: 190; TrLCRa5: SEQ ID NO: 191; TrLCRa6: SEQ ID NO: 192; TrLCRa7: SEQ ID NO: 193).
FIG. 68 shows the nucleotide sequence of TrF3′5′Ha (SEQ ID NO: 194).
FIG. 69 shows the deduced amino acid sequence of TrF3′5′Ha (SEQ ID NO: 195).
FIG. 70 shows the consensus contig nucleotide sequence of TrF3′5′Hb (SEQ ID NO: 196).
FIG. 71 shows the deduced amino acid sequence of TrF3′5′Hb (SEQ ID NO: 197).
FIG. 72A and FIG. 72B show the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence TrF3′5′Hb (TrF3′5′Hb1: SEQ ID NO: 198; TrF3′5′Hb2: SEQ ID NO: 199; TrF3′5′Hb3: SEQ ID NO: 200; TrF3′5′Hb4: SEQ ID NO: 201).
FIG. 73A and FIG. 73B show the consensus contig nucleotide sequence of TrF3Ha (SEQ ID NO: 202).
FIG. 74 shows the deduced amino acid sequence of TrF3Ha (SEQ ID NO: 203).
FIG. 75A-FIG. 75V show the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence TrF3Ha (TrF3Ha1: SEQ ID NO: 204; TrF3Ha2: SEQ ID NO: 205; TrF3Ha3: SEQ ID NO: 206; TrF3Ha4: SEQ ID NO: 207; TrF3Ha5: SEQ ID NO: 208; TrF3Ha6: SEQ ID NO: 209; TrF3Ha7: SEQ ID NO: 210; TrF3Ha8: SEQ ID NO: 211; TrF3Ha9: SEQ ID NO: 212; TrF3Ha10: SEQ ID NO: 213; TrF3Ha11: SEQ ID NO: 214; TrF3Ha12: SEQ ID NO: 215; TrF3Ha13: SEQ ID NO: 216; TrF3Ha14: SEQ ID NO: 217; TrF3Ha15: SEQ ID NO: 218; TrF3Ha16: SEQ ID NO: 219; TrF3Ha17: SEQ ID NO: 220; TrF3Ha18: SEQ ID NO: 221; TrF3Ha19: SEQ ID NO: 222; TrF3Ha20: SEQ ID NO: 223; TrF3Ha21: SEQ ID NO: 224; TrF3Ha22: SEQ ID NO: 225; TrF3Ha23: SEQ ID NO: 226; TrF3Ha24: SEQ ID NO: 227; TrF3Ha25: SEQ ID NO: 228; TrF3Ha26: SEQ ID NO: 229; TrF3Ha27: SEQ ID NO: 230; TrF3Ha28: SEQ ID NO: 231; TrF3Ha29: SEQ ID NO: 232; TrF3Ha30: SEQ ID NO: 233; TrF3Ha31: SEQ ID NO: 234; TrF3Ha32: SEQ ID NO: 235; TrF3Ha33: SEQ ID NO: 236; TrF3Ha34: SEQ ID NO: 237; TrF3Ha35: SEQ ID NO: 238; TrF3Ha36: SEQ ID NO: 239; TrF3Ha37: SEQ ID NO: 240; TrF3Ha38: SEQ ID NO: 241; TrF3Ha39: SEQ ID NO: 242; TrF3Ha40: SEQ ID NO: 243; TrF3Ha41: SEQ ID NO: 244).
FIG. 76 shows the nucleotide sequence of TrF3Hb (SEQ ID NO: 245).
FIG. 77 shows the deduced amino acid sequence of TrF3Hb (SEQ ID NO: 246).
FIG. 78 shows the nucleotide sequence of TrF3Hc (SEQ ID NO: 247).
FIG. 79 shows the deduced amino acid sequence of TrF3Hc (SEQ ID NO: 248).
FIG. 80 shows the consensus contig nucleotide sequence of TrF3′Ha (SEQ ID NO: 249).
FIG. 81 shows the deduced amino acid sequence of TrF3′Ha (SEQ ID NO: 250).
FIG. 82 shows the nucleotide sequences of the nucleic acid: fragments contributing to the consensus contig sequence TrF3′Ha (TrF3′Ha1: SEQ ID NO: 251; TrF3′1Ha2: SEQ ID NO: 252).
FIG. 83 shows the consensus contig nucleotide sequence of TrPALa (SEQ ID NO: 253).
FIG. 84 shows the deduced amino acid sequence of TrPALa (SEQ ID NO: 254).
FIG. 85 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence TrPALa (TrPALa1: SEQ ID NO: 255; TrPALa2: SEQ ID NO: 256; TrPALa3: SEQ ID NO: 257).
FIG. 86 shows the consensus contig nucleotide sequence of TrPALb (SEQ ID NO: 258).
FIG. 87 shows the deduced amino acid sequence of TrPALb (SEQ ID NO: 259).
FIG. 88A and FIG. 88B show the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence TrPALb (TrPALb1: SEQ ID NO: 260; TrPALb2: SEQ ID NO: 261; TrPALb3: SEQ ID NO: 262; TrPALb4: SEQ ID NO: 263; TrPALb5: SEQ ID NO: 264; TrPALb6: SEQ ID NO: 265; TrPALb7: SEQ ID NO: 266; TrPALb8: SEQ ID NO: 267).
FIG. 89 shows the nucleotide sequence of TrPALc (SEQ ID NO: 268).
FIG. 90 shows the deduced amino acid sequence of TrPALc (SEQ ID NO: 269).
FIG. 91 shows the nucleotide sequence of TrPALd (SEQ ID NO: 270).
FIG. 92 shows the deduced amino acid sequence of TrPALd (SEQ ID NO: 271).
FIG. 93 shows the nucleotide sequence of TrPALe (SEQ ID NO: 272).
FIG. 94 shows the deduced amino acid sequence of TrPALe (SEQ ID NO: 273).
FIG. 95 shows the consensus contig nucleotide sequence of TrPALf (SEQ ID NO: 274).
FIG. 96 shows the deduced amino acid sequence of TrPALf (SEQ ID NO: 275).
FIG. 97 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence TrPALf (TrPALf1: SEQ ID NO: 276; TrPALf2: SEQ ID NO: 277).
FIG. 98 shows the consensus contig nucleotide sequence of TrVRa (SEQ ID NO: 278).
FIG. 99 shows the deduced amino acid sequence of TrVRa (SEQ ID NO: 279).
FIG. 100A-FIG. 100C show the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence TrVRa (TrVRa1: SEQ ID NO: 280; TrVRa2: SEQ ID NO: 281; TrVRa3: SEQ ID NO: 282; TrVRa4: SEQ ID NO: 283; TrVRa5: SEQ ID NO: 284; TrVRa6: SEQ ID NO: 285).
FIG. 101 shows the consensus contig nucleotide sequence of LpDFRa (SEQ ID NO: 286).
FIG. 102 shows the deduced amino acid sequence of LpDFRa (SEQ ID NO: 287).
FIG. 103A-FIG. 103B show the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpDFRa (LpDFRa1: SEQ ID NO: 288; LpDFRa2: SEQ ID NO: 289; LpDFRa3: SEQ ID NO: 290; LpDFRa4: SEQ ID NO: 291; LpDFRa5: SEQ ID NO: 292).
FIG. 104 shows the consensus contig nucleotide sequence of LpDFRb (SEQ ID NO: 293).
FIG. 105 shows the deduced amino acid sequence of LpDFRb (SEQ ID NO: 294).
FIG. 106 shows the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpDFRb (LpDFRb1: SEQ ID NO: 295; LpDFRb2: SEQ ID NO: 296; LpDFRb3: SEQ ID NO: 297).
FIG. 107A-FIG. 107B show the consensus contig nucleotide sequence of LpF3Ha (SEQ ID NO: 298).
FIG. 108 shows the deduced amino acid sequence of LpF3Ha (SEQ ID NO: 299).
FIG. 109A-FIG. 109B show the nucleotide sequences of the nucleic acid fragments contributing to the consensus contig sequence LpF3Ha (LpF3Ha1: SEQ ID NO: 300; LpF3Ha2: SEQ ID NO: 301; LpF3Ha3: SEQ ID NO: 302).
FIG. 110 shows a plasmid map of the cDNA encoding perennial ryegrass F3OH.
FIG. 111 shows the full nucleotide sequence of perennial ryegrass F3OH cDNA (SEQ ID NO: 303).
FIG. 112 shows the deduced amino acid sequence of perennial ryegrass F3OH cDNA (SEQ ID NO: 304).
FIG. 113 shows plasmid maps of sense and antisense constructs of LpF3OH in pDH51 transformation vector.
FIG. 114 shows plasmid maps of sense and antisense constructs of LpF3OH in pPZP221:35S2 binary transformation-vector.
FIG. 115 shows screening by Southern hybridization for RFLPs using LpF3OH as a probe.
FIG. 116 shows a plasmid map of the cDNA encoding white clover BANa.
FIG. 117 shows the full nucleotide sequence of white clover BANa cDNA (SEQ ID NO: 305).
FIG. 118 shows the deduced amino acid sequence of white clover BANa cDNA (SEQ ID NO: 306).
FIG. 119 shows plasmid maps of sense and antisense constructs of TrBANa in pDH51 transformation vector.
FIG. 120 shows plasmid maps of sense and antisense constructs of TrBANa in pPZP221:35S2 binary transformation vector.
FIG. 121 shows a plasmid map of the cDNA encoding white clover CHIa.
FIG. 122 shows the full nucleotide sequence of white clover CHIa cDNA (SEQ ID NO: 307).
FIG. 123 shows the deduced amino acid sequence of white clover CHIa cDNA (SEQ ID NO: 308).
FIG. 124 shows plasmid maps of sense and antisense constructs of TrCHIa in pDH51 transformation vector.
FIG. 125 shows plasmid maps of sense and antisense constructs of TrCHIa in pPZP221:35S2 binary transformation vector.
FIG. 126 shows a plasmid map of the cDNA encoding white clover CHId.
FIG. 127 shows the full nucleotide sequence of white clover CHId cDNA (SEQ ID NO: 309).
FIG. 128 shows the deduced amino acid sequence of white clover CHId cDNA (SEQ ID NO: 310).
FIG. 129 shows plasmid maps of sense and antisense constructs of TrCHId in pDH51 transformation vector.
FIG. 130 shows plasmid maps of sense and antisense constructs of TrCHId in pPZP221:35S2 binary transformation vector.
FIG. 131 shows a plasmid map of the cDNA encoding white clover CHRc.
FIG. 132 shows the full nucleotide sequence of white clover CHRc cDNA (SEQ ID NO: 311).
FIG. 133 shows the deduced amino acid sequence of white clover CHRc cDNA (SEQ ID NO: 312).
FIG. 134 shows plasmid maps of sense and antisense constructs of TrCHRc in pDH51 transformation vector.
FIG. 135 shows plasmid maps of sense and antisense constructs of TrCHRc in pPZP221:35S2 binary transformation vector.
FIG. 136 shows a plasmid map of the cDNA encoding white clover CHSa1.
FIG. 137 shows the full nucleotide sequence of white clover CHSa1 cDNA (SEQ ID NO: 313).
FIG. 138 shows the deduced amino acid sequence of white clover CHSa1 cDNA (SEQ ID NO: 314).
FIG. 139 shows plasmid maps of sense and antisense constructs of TrCHSa1 in pDH51 transformation vector.
FIG. 140 shows plasmid maps of sense and antisense constructs of TrCHSa1 in pPZP221:35S2 binary transformation vector.
FIG. 141 shows a plasmid map of the cDNA encoding white clover CHSa3.
FIG. 142 shows the full nucleotide sequence of white clover CHSa3 cDNA (SEQ ID NO: 315).
FIG. 143 shows the deduced amino acid sequence of white clover CHSa3 cDNA (SEQ ID NO: 316).
FIG. 144 shows plasmid maps of sense and antisense constructs of TrCHSa3 in pDH51 transformation vector.
FIG. 145 shows plasmid maps of sense and antisense constructs of TrCHSa3 in pPZP221:35S2 binary transformation vector.
FIG. 146 shows a plasmid map of the cDNA encoding white clover CHSc.
FIG. 147 shows the full nucleotide sequence of white clover CHSc cDNA (SEQ ID NO: 317).
FIG. 148 shows the deduced amino acid sequence of white clover CHSc cDNA (SEQ ID NO: 318).
FIG. 149 shows plasmid maps of sense and antisense constructs of TrCHSc in pDH51 transformation vector.
FIG. 150 shows plasmid maps of sense and antisense constructs of TrCHSc in pPZP221:35S2 binary transformation vector.
FIG. 151 shows a plasmid map of the cDNA encoding white clover CHSd2.
FIG. 152 shows the full nucleotide sequence of white clover CHSd2 cDNA (SEQ ID NO: 319).
FIG. 153 shows the deduced amino acid sequence of white clover CHSd2 cDNA (SEQ ID NO: 320).
FIG. 154 shows plasmid maps of sense and antisense constructs of TrCHSd2 in pDH51 transformation vector.
FIG. 155 shows plasmid maps of sense and antisense constructs of TrCHSd2 in pPZP221:35S2 binary transformation vector.
FIG. 156 shows a plasmid map of the cDNA encoding white clover CHSf.
FIG. 157 shows the full nucleotide sequence of white clover CHSf cDNA (SEQ ID NO: 321).
FIG. 158 shows the deduced amino acid sequence of white clover CHSf cDNA (SEQ ID NO: 322).
FIG. 159 shows plasmid maps of sense and antisense constructs of TrCHSf in pDH51 transformation vector.
FIG. 160 shows plasmid maps of sense and antisense constructs of TrCHSf in pPZP221:35S2 binary transformation vector.
FIG. 161 shows a plasmid map of the cDNA encoding white clover CHSh.
FIG. 162 shows the full nucleotide sequence of white clover CHSh cDNA (SEQ ID NO: 323).
FIG. 163 shows the deduced amino acid sequence of white clover CHSh cDNA (SEQ ID NO: 324).
FIG. 164 shows plasmid maps of sense and antisense constructs of TrCHSh in pDH51 transformation vector.
FIG. 165 shows plasmid maps of sense and antisense constructs of TrCHSh in pPZP221:35S2 binary transformation vector.
FIG. 166 shows a plasmid map of the cDNA encoding white clover DFRd.
FIG. 167 shows the full nucleotide sequence of white clover DFRd cDNA (SEQ ID NO: 325).
FIG. 168 shows the deduced amino acid sequence of white clover DFRd cDNA (SEQ ID NO: 326).
FIG. 169 shows plasmid maps of sense and antisense constructs of TrDFRd in pDH51 transformation vector.
FIG. 170 shows plasmid maps of sense and antisense constructs of TrDFRd in pPZP221:35S2 binary transformation vector.
FIG. 171 shows a plasmid map of the cDNA encoding white clover F3Ha.
FIG. 172 shows the full nucleotide sequence of white clover F3Ha cDNA (SEQ ID NO: 327).
FIG. 173 shows the deduced amino acid sequence of white clover F3Ha cDNA (SEQ ID NO: 328).
FIG. 174 shows plasmid maps of sense and antisense constructs of TrF3Ha in pDH51 transformation vector.
FIG. 175 shows plasmid maps of sense and antisense constructs of TrF3Ha in pPZP221:35S2 binary transformation vector.
FIG. 176 shows a plasmid map of the cDNA encoding white clover PALa.
FIG. 177 shows the full nucleotide sequence of white clover PALa cDNA (SEQ ID NO: 329).
FIG. 178 shows the deduced amino acid sequence of white clover PALa cDNA (SEQ ID NO: 330).
FIG. 179 shows plasmid maps of sense and antisense constructs of TrPALa in pDH51 transformation vector.
FIG. 180 shows plasmid maps of sense and antisense constructs of TrPALa in pPZP221:35S2 binary transformation vector.
FIG. 181 shows a plasmid map of the cDNA encoding white clover PALb.
FIG. 182 shows the full nucleotide sequence of white clover PALb cDNA (SEQ ID NO: 331).
FIG. 183 shows the deduced amino acid sequence of white clover PALb cDNA (SEQ ID NO: 332).
FIG. 184 shows plasmid maps of sense and antisense constructs of TrPALb in pDH51 transformation vector.
FIG. 185 shows plasmid maps of sense and antisense constructs of TrPALb in pPZP221:35S2 binary transformation vector.
FIG. 186 shows a plasmid map of the cDNA encoding white clover PALf.
FIG. 187 shows the full nucleotide sequence of white clover PALf cDNA (SEQ ID NO: 333).
FIG. 188 shows the deduced amino acid sequence of white clover PALf cDNA (SEQ ID NO: 334).
FIG. 189 shows plasmid maps of sense and antisense constructs of TrPALf in pDH51 transformation vector.
FIG. 190 shows plasmid maps of sense and antisense constructs of TrPALf in pPZP221:35S2 binary transformation vector.
FIG. 191 shows a plasmid map of the cDNA encoding white clover VRa.
FIG. 192 shows the full nucleotide sequence of white clover VRa cDNA (SEQ ID NO: 335).
FIG. 193 shows the deduced amino acid sequence of white clover VRa cDNA (SEQ ID NO: 336).
FIG. 194 shows plasmid maps of sense and antisense constructs of TrVRa in pDH51 transformation vector.
FIG. 195 shows plasmid maps of sense and antisense constructs of TrVRa in pPZP221:35S2 binary transformation vector.
FIG. 196A and FIG. 196B show (A), infiltration of Arabidopsis plants; (B), selection of transgenic Arabidopsis plants on medium containing 75 μg/ml gentamycin; (C), young transgenic Arabidopsis plants; (D) and (E), two representative results of real-time PCR analysis of Arabidopsis transformed with chimeric genes involved in flavonoid biosynthesis.
FIG. 197 shows the genetic map detailing the relation of perennial ryegrass genes involved in flavonoid biosynthesis.



DETAILED DESCRIPTION OF THE INVENTION
Example 1
Preparation of cDNA Libraries, Isolation and Sequencing of cDNAS Coding for CHI, CHI-Like, CHS, CHS-Like, CHR, CHR-Like, DFR, DFR-Like, LCR, LCR-Like, F3′5′H, F3′5′H-Like, F3H, F3H-Like, F3′H, F3′H-Like, PAL, PAL-Like, VR and VR-Like Proteins from White Clover (Trifolium repens) and Perennial Ryegrass (Lolium perenne)
cDNA libraries representing mRNAs from various organs and tissues of white clover (Trifolium repens) and perennial ryegrass (Lolium perenne) were prepared. The characteristics of the white clover and perennial ryegrass libraries, respectively, are described below (Tables 1 and 2).







TABLE 1







cDNA LIBRARIES FROM WHITE CLOVER (Trifolium repens)








Library
Organ/Tissue





01wc
Whole seedling, light grown


02wc
Nodulated root 3, 5, 10, 14, 21 & 28 day old seedling


03wc
Nodules pinched off roots of 42 day old rhizobium inoculated



plants


04wc
Cut leaf and stem collected after 0, 1, 4, 6 & 14 h after cutting


05wc
Inflorescences: <50% open, not fully open and fully open


06wc
Dark grown etiolated


07wc
Inflorescence - very early stages, stem elongation, <15 petals,



15-20 petals


08wc
seed frozen at −80° C., imbibed in dark overnight at 10° C.


09wc
Drought stressed plants


10wc
AMV infected leaf


11wc
WCMV infected leaf


12wc
Phosphorus starved plants


13wc
Vegetative stolon tip


14wc
stolon root initials


15wc
Senescing stolon


16wc
Senescing leaf
















TABLE 2







cDNA LIBRARIES FROM PERENNIAL RYEGRASS


(Lolium perenne)








Library
Organ/Tissue





01rg
Roots from 3-4 day old light-grown seedlings


02rg
Leaves from 3-4 day old light-grown seedlings


03rg
Etiolated 3-4 day old dark-grown seedlings


04rg
Whole etiolated seedlings (1-5 day old and 17 days old)


05rg
Senescing leaves from mature plants


06rg
Whole etiolated seedlings (1-5 day old and 17 days old)


07rg
Roots from mature plants grown in hydroponic culture


08rg
Senescent leaf tissue


09rg
Whole tillers and sliced leaves (0, 1, 3, 6, 12 and 24 h after



harvesting)


10rg
Embryogenic suspension-cultured cells


11rg
Non-embryogenic suspension-cultured cells


12rg
Whole tillers and sliced leaves (0, 1, 3, 6, 12 and 24 h after



harvesting)


13rg
Shoot apices including vegetative apical meristems


14rg
Immature inflorescences including different stages of inflores-



cence meristem and inflorescence development


15rg
Defatted pollen


16rg
Leaf blades and leaf sheaths (rbcL, rbcS, cab, wir2A subtracted)


17rg
Senescing leaves and tillers


18rg
Drought-stressed tillers (pseudostems from plants subjected to



PEG-simulated drought stress)


19rg
Non-embryogenic suspension-cultured cells subjected to osmotic



stress (grown in media with half-strength salts)



(1, 2, 3, 4, 5, 6, 24 and 48 h after transfer)


20rg
Non-embryogenic suspension-cultured cells subjected to osmotic



stress (grown in media with double-strength salts)



(1, 2, 3, 4, 5, 6, 24 and 48 h after transfer)


21rg
Drought-stressed tillers (pseudostems from plants subjected to



PEG-simulated drought stress)


22rg
Spikelets with open and maturing florets


23rg
Mature roots (specific subtraction with leaf tissue)









The cDNA libraries may be prepared by any of many methods available. For example, total RNA may be isolated using the TRIZOL method (Gibco-BRL; USA) or the RNEASY Plant Mini kit (Qiagen; Germany), following the manufacturers′ instructions. cDNAs may be generated using the SMART PCR cDNA synthesis kit (Clontech; USA), cDNAs may be amplified by long distance polymerase chain reaction using the ADVANTAGE 2 PCR Enzyme system (Clontech; USA), cDNAs may be cleaned using the GENECLEAN spin column (Bio 101; USA), tailed and size fractionated, according to the protocol provided by Clontech. The cDNAs may be introduced into the pGEM-T Easy Vector system 1 (Promega; USA) according to the protocol provided by Promega. The cDNAs in the pGEM-T Easy plasmid vector are transfected into Escherichia coli Epicurian coli XL10-Gold ultra competent cells (Stratagene, USA) according to the protocol provided by Stratagene.
Alternatively, the cDNAs may be introduced into plasmid vectors for first preparing the cDNA libraries in Uni-ZAP XR vectors according to the manufacturer's protocol (Stratagene Cloning Systems; La Jolla, Calif., USA). The Uni-ZAP XR libraries are converted into plasmid libraries according to the protocol provided by Stratagene. Upon conversion, cDNA inserts will be contained in the plasmid vector pBluescript. In addition, the cDNAs may be introduced directly into precut pBluescript II SK(+) vectors (Stratagene) using T4 DNA ligase (New England Biolabs), followed by transfection into E. coli DH10B cells according to the manufacturer's protocol (GIBCO BRL Products).
Once the cDNA inserts are in plasmid vectors, plasmid DNAs were prepared from randomly picked bacterial colonies containing recombinant plasmids, or the insert cDNA sequences were amplified via polymerase chain reaction using primers specific for vector sequences flanking the inserted cDNA sequences. Plasmid DNA preparation was performed robotically using the Qiagen QiaPrep Turbo kit (Qiagen; Germany) according to the protocol provided by Qiagen. Amplified insert DNAs were sequenced in dye-terminator sequencing reactions to generate partial cDNA sequences (expressed sequence tags or “ESTs”). The resulting ESTs were analyzed using an Applied Biosystems ABI 3700 sequence analyser.
Example 2
DNA Sequence Analyses
The cDNA clones encoding CHI, CHI-like, CHS, CHS-like, CHR, CHR-like, DFR, DFR-like, LCR, LCR-like, F3′5′H, F3′5′H-like, F3H, F3H-like, F3′H, F3′H-like, PAL, PAL-like, VR and VR-like proteins were identified by conducting BLAST (Basic Local Alignment Search Tool; Altschul et al. (1993) J. Mol. Biol. 215:403-410) searches. The cDNA sequences obtained were analyzed for similarity to all publicly available DNA sequences contained in the eBioinformatics nucleotide database using the BLASTN algorithm provided by the National Center for Biotechnology Information (NCBI). The DNA sequences were translated in all reading frames and compared for similarity to all publicly available protein sequences contained in the SWISS-PROT protein sequence database using BLASTx algorithm (v 2.0.1) (Gish and States (1993) Nature Genetics 3:266-272) provided by the NCBI.
The cDNA sequences obtained and identified were then used to identify additional identical and/or overlapping cDNA sequences generated using the BLASTN algorithm. The identical and/or overlapping sequences were subjected to a multiple alignment using the CLUSTALw algorithm, and to generate a consensus contig sequence derived from this multiple sequence alignment. The consensus contig sequence was then used as a query for a search against the SWISS-PROT protein sequence database using the BLASTx algorithm to confirm the initial identification.
Example 3
Identification and Full-Length Sequencing of cDNAS Encoding Perennial Ryegrass F3OH and White Clover BANa, CHIa, CHId, CHRc, CHSa1, CHSa3, CHSc, CHSd2, CHSf, CHSh, DFRd, F3Ha, PALa, PALb, PALf and VRa Proteins
To fully characterize for the purposes of the generation of probes for hybridization experiments and the generation of transformation vectors, a set of cDNAs encoding perennial ryegrass F3OH and white clover BANa, CHIa, CHId, CHRc, CHSa1, CHSa3, CHSc, CHSd2, CHSf, CHSh, DFRd, F3Ha, PALa, PALb, PALf and VRa proteins was identified and fully sequenced.
Full-length cDNAs were identified from our EST sequence database using relevant published sequences (NCBI databank) as queries for BLAST searches. Full-length cDNAs were identified by alignment of the query and hit sequences using SEQUENCHER (Gene Codes Corp.; Ann Arbor, Mich.). The original plasmid was then used to transform chemically competent XL-1 cells (prepared in-house, CaCl2 protocol). After colony PCR (using HotStarTaq, Qiagen), a minimum of three PCR-positive colonies per transformation were picked for initial sequencing with M13F and M13R primers. The resulting sequences were aligned with the original EST sequence using Sequencher to confirm the identity. One of the three clones having the best initial sequencing result was picked for full-length sequencing.
Sequencing was completed by primer walking, i.e. oligonucleotide primers were designed to the initial sequence and used for further sequencing. In most cases, the sequencing could be done from both 5′ and 3′ end. The sequences of the oligonucleotide primers are shown in Table 3. In some instances, however, an extended poly-A tail necessitated the sequencing of the cDNA to be completed from the 5′ end.
Contigs were then assembled in Sequencher. The contigs include the sequences of the SMART primers used to generate the initial cDNA library as well as pGEM-T Easy vector sequence up to the EcoRI cut site both at the 5′ and 3′ end.
Plasmid maps and the full cDNA sequences of perennial ryegrass F3OH and white clover BANa, CHIa, CHId, CHRc, CHSa1, CHSa3, CHSc, CHSd2, CHSf, CHSh, DFRd, F3Ha, PALa, PALb, PALf and VRa proteins were obtained (FIGS. 110, 116, 121, 126, 131, 136, 141, 146, 151, 156, 161, 166, 171, 176, 181, 186 and 191).








TABLE 3







LIST OF PRIMERS USED FOR SEQUENCING OF THE



FULL-LENGTH cDNAS


 












GENE

SEQUENCING
PRIMER SEQUENCE 
SEQ ID



NAME
CLONE ID
PRIMER
(5′ > 3′)
NO





LpF3OH
08rg1YsF07
08rg1YsF07.f1
TTGAGAGCTTCGTCGACC
337



 




08rg1YsF07.r1
AACTCCTCGTAGTACTCC
338


 


TrCHRc
11wc1IsD03
11wc1IsD03.f1
TTCAATTGGAGTACTTGG
339


 




11wc1IsD03.r1
ACTCCTTGTTCATATAACC
340


 


TrCHSa1
02wc2FsD07
02wc2FsD07.f1
ACATGGTGGTGGTTGAGG
341


 




02wc2FsD07.f2
TGCTGCACTCATTGTTGG
342

















TABLE 3







LIST OF PRIMERS USED FOR SEQUENCING OF THE



FULL-LENGTH cDNAS


 












GENE

SEQUENCING
PRIMER SEQUENCE
SEQ ID



NAME
CLONE ID
PRIMER
(5′ > 3′)
NO







02wc2FsD07.f3
ACATTGATAAGGCATTGG
343



 


TrCHSa3
05wc1RsB06
05wc1RsB06.f1
AGGAGGCTGCAGTCAAGG
344


 




05wc1RsB06.f2
TGCCTGAAATTGAGAAACC
345


 




05wc1RsB06.f3
AAAGCTAGCCTTGAAGCC
346


 


TrCHSc
07wc1TsE12
07wc1TsE12.f1
TCGGACATAACTCATGTGG
347


 




07wc1TsE12.f2
TTGGGTTGGAGAATAAGG
348


 




07wc1TsE12.r1
TGGACATTTATTGGTTGC
349


 




07wc1TsE12.r2
TATCATGTCTGGAAATGC
350


 


TrCHSd2
07wc1XsD03
07wc1XsD03.f1
TTTATGTGAGTACATGGC
351


 




07wc1XsD03.f2
AGCAGCTGTGATTGTAGG
352


 




07wc1XsD03.f3
TGAGAAAGCTCTTGTTGAGG
353


 


TrCHSf
07wc1UsD07
07wc1UsD07.f1
AGATTGCATCAAAGAATGG
354


 




07wc1UsD07.r1
GGTCCAAAAGCCAATCC
355


 


TrCHSh
13wc2IsG04
13wc2IsG04.f1
TAAGACGAGACATAGTGG
356


 




13wc2IsG04.r1
TATTCACTAAGCACATGC
357


 


TrDFRd
12wc1CsE09
12wc1CsE09.f1
TTACCTCGTCTGTCTCG
358


 




12wc1CsE09.r1
AACACACACATGTCTACC
359


 


TrF3Ha
07wc1LsG03
07wc1LsG03.f1
TGAAGGATTGGAGAGAGC
360


 




07wc1LsG03.r1
TACACAGTTGCATCTGG
361


 


TrPALa
04wc1UsB03
04wc1UsB03.f1
ATCGGAATCTGCTAGAGC
362


 




04wc1UsB03.f2
TGTTGGTTCTGGTTTAGC
363


 




04wc1UsB03.r1
TTCATATGCAATCCTTGC
364


 




04wc1UsB03.r2
TCTTGGTTGTGTTGTTCC
365


 


TrPALb
05wc1PsH02
05wc1PsH02.f1
TGGGACTGATAGTTATGG
366


 




05wc1PsH02.f2
TCTTGCTCTTGTTAATGG
367


 




05wc1PsH02.r1
AGCACCATTCCACTCTCC
368


 




05wc1PsH02.r2
TTCTCTTCGCTACTTGGC
369


 


TrPALf
13wc2AsD12
13wc2AsD12.f1
ATAGTGGTGTGAGGGTGG
370


 




13wc2AsD12.f2
TCTTGTTAATGGTACTGC
371


 




13wc2AsD12.r1
ATTTATCGCACTCTTCGC
372


 




13wc2AsD12.r2
AAAGTGGAAGACATGAGC
373


 


TrVRa
11wc1NsA07
11wc1NsA07.f1
AAGAACAGTGGATGGAGC
374


 




11wc1NsA07.r1
TCAACTCATCTACTGATAG
375









Example 4
Development of Transformation Vectors Containing Chimeric Genes with cDNA Sequences from Perennial Ryegrass F3OH and White Clover BANa, CHIa, CHId, CHRc, CHSa1, CHSa3, CHSc, CHSd2, CHSf, CHSh, DFRd, F3Ha, PALa, PALb, PALf and VRa
To alter the expression of the proteins involved in flavonoid biosynthesis, protein binding, metal chelation, anti-oxidation, UV-light absorption, tolerance to biotic stresses such as viruses, micro-organisms, insects and fungal pathogens; pigmentation in for example flowers and leaves; herbage quality and bloat-safety and isoflavonoid content leading to health benefits, perennial ryegrass F3OH and white clover BANa, CHIa, CHId, CHRc, CHSa1, CHSa3, CHSc, CHSd2, CHSf, CHSh, DFRd, F3Ha, PALa, PALb, PALf and VRa, through antisense and/or sense suppression technology and for over-expression of these key enzymes in transgenic plants, a set of sense and antisense transformation vectors was produced.
cDNA fragments were generated by high fidelity PCR using the original pGEM-T Easy plasmid cDNA as a template. The primers used (Table 4) contained recognition sites for appropriate restriction enzymes, for example EcoRI and XbaI, for directional and non-directional cloning into the target vector. After PCR amplification and restriction digest with the appropriate restriction enzyme (usually XbaI), the cDNA fragments were cloned into the corresponding site in pDH51, a pUC18-based transformation vector containing a CaMV 35S expression cassette. The orientation of the constructs (sense or antisense) was checked by DNA sequencing through the multi-cloning site of the vector. Transformation vectors containing chimeric genes using full-length open reading frame cDNAs encoding perennial ryegrass F3OH and white clover BANa, CHIa, CHId, CHRc, CHSa1, CHSa3, CHSc, CHSd2, CHSf, CHSh, DFRd, F3Ha, PALa, PALb, PALf and VRa proteins in sense and antisense orientations under the control of the CaMV 35S promoter were generated (FIGS. 113, 119, 124, 129, 134, 139, 144, 149, 154, 159, 164, 169, 174, 179, 184, 189 and 194).







TABLE 4







LIST OF PRIMERS USED TO PCR-AMPLIFY THE OPEN READING FRAMES


 












GENE


PRIMER SEQUENCE
SEQ ID



NAME
CLONE ID
PRIMER
(5′->3′)
NO





LpF3OH
08rg1YsF07
08rg1YsF07f
GAATTCTAGAAGCAGAAAGTACGGACATCAGC
376



 




08rg1YsF07r
GAATTCTAGAACCATATGGCGACACATCG
377


 


TrBANa
05wc2XsG02
05wc2XsG02f
GGATCCTCTAGAGCACTAGTGTGTATAAGTTTCTTGG
378


 




05wc2XsG02r
GGATCCTCTAGACCCCCTTAGTCTTAAAATACTCG
379


 


TrCHIa
06wc2AsF12
06wc2AsF12f
GAATTCTAGAGATCTGAAACAACATAGTCACC
380


 




06wc2AsF12r
GAATTCTAGATCAATCTTGTGCTGCAATGC
381


 


TrCHId
12wc1FsG04
12wc1FsG04f
GAATTCTAGAAAGTTCAACGAGATCAATGG
382


 




12wc1FsG04r
GAATTCTAGATTCCGCTTGGTCTTTATTGC
383


 


TrCHRc
11wc1IsD03
11wc1IsD03f
GAATTCTAGAACATGGGTAGTGTTGAAATTCC
384


 




11wc1IsD03r
GAATTCTAGAAGATATTGAGTGAGCTTAAGG
385


 


TrCHSa1
02wc2FsD07
02wc2FsD07f
GACGTCGACATTACATACATAGCAGGAAC
386


 




02wc2FsD07r
GACGTCGACAGTCTCTCATTCTCATATAGC
387


 


TrCHSa3
05wc1RsB06
05wc1RsB06f
GAATTCTAGAAGATATGGTGAGTGTAGCTG
388


 




05wc1RsB06r
GAATTCTAGAATCACACATCTTATATAGCC
389


 


TrCHSc
07wc1TsE12
07wc1TsE12f
GAATTCTAGAAGAAGAAATATGGGAGACGAAGG
390


 




07wc1TsE12r
GAATTCTAGAAAGACTTCATGCACACAAGTTCC
391


 


TrCHSd2
07wc1XsD03
07wc1XsD03f
GAATTCTAGAATAACCTATCAGTACTCACC
392


 




07wc1XsD03r
GAATTCTAGAATCTAGGCAATTTAAGTGGC
393


 


TrCHSf
07wc1UsD07
07wc1UsD07f
GAATTCTAGATGATTCATTGTTTGTTTCCATAAC
394


 




07wc1UsD07r
GAATTCTAGAACATATTCATCTTCCTATCAC
395


 


TrCHSh
13wc2IsG04
13wc2IsG04f
GAATTCTAGATCCAAATTCTCGTACCTCACC
396


 




13wc2IsG04r
GAATTCTAGATAGTTCACATCTCTCGGCAGG
397


 


TrDFRd
12wc1CsE09
12wc1CsE09f
GACGTCGACACAACAGTCTTCCACTTGAGC
398


 




12wc1CsE09r
GACGTCGACTCTATACTCTGGTAACTATAGG
399


 


TrF3Ha
07wc1LsG03
07wc1LsG03f
GAATTCTAGAACCACACAACACACAAACACC
400


 




07wc1LsG03r
GAATTCTAGAACCAAGCAGCTTAATACACG
401


 


TrPALa
04wc1UsB03
04wc1UsB03f
AGTACTGCAGAGATATGGAAGTAGTAGCAGCAGC
402


 




04wc1UsB03r
AGTACTGCAGTAGCAAACCAGTTCCCAACTCC
403


 


TrPALb
05wc1PsH02
05wc1PsH02f
AGTACTGCAGATAATGGAGGGAATTACCAATGG
404


 




05wc1PsH02r
AGTACTGCAGTGCTAATTAACATATTGGTAGAGG
405


 


TrPALf
13wc2AsD12
13wc2AsD12f
AGTACTGCAGATAATGGAGGGAATTACCAATGG
406


 




13wc2AsD12r
AGTACTGCAGTGCTAATTAACATATTGGTAGAGG
407


 


TrVRa
11wc1NsA07
11wc1NsA07f
AGTACTGCAGATAAAGAGAGTCAAAAATGGC
408


 




11wc1NsA07r
AGTACTGCAGAACACATACTTAGAGATAGCC
409









Example 5
Development of Binary Transformation Vectors Containing Chimeric Genes with cDNA Sequences from Perennial Ryegrass F3OH and White Clover BANa, CHIa, CHId, CHRc, CHSa1, CHSa3, CHSc, CHSd2, CHSf, CHSh, DFRd, F3Ha, PALa, PALb, PALf and VRa
To alter the expression of the proteins involved in flavonoid biosynthesis, protein binding, metal chelation, anti-oxidation, UV-light absorption, tolerance to biotic stresses such as viruses, micro-organisms, insects and fungal pathogens; pigmentation in for example flowers and leaves; herbage quality and bloat-safety and isoflavonoid content leading to health benefits, perennial ryegrass F3OH and white clover BANa, CHIa, CHId, CHRc, CHSa1, CHSa3, CHSc, CHSd2, CHSf, CHSh, DFRd, F3Ha, PALa, PALb, PALf and VRa, through antisense and/or sense suppression technology and for over-expression of these key proteins in transgenic plants, a set of sense and antisense binary transformation vectors was produced.
cDNA fragments were generated by high fidelity PCR using the original pGEM-T Easy plasmid cDNA as a template. The primers used (Table 4) contained recognition sites for appropriate restriction enzymes, for example EcoRI and XbaI, for directional and non-directional cloning into the target vector. After PCR amplification and restriction digest with the appropriate restriction enzyme (usually XbaI), the cDNA fragments were cloned into the corresponding site in a modified pPZP binary vector (Hajdukiewicz et al., 1994). The pPZP221 vector was modified to contain the 35S2 cassette from pKYLX71:35S2 as follows. pKYLX71:35S2 was cut with ClaI. The 5′ overhang was filled in using Klenow and the blunt end was A-tailed with Taq polymerase. After cutting with EcoRI, the 2 kb fragment with an EcoRI-compatible and a 3′-A tail was gel-purified. pPZP221 was cut with HindIII and the resulting 5′ overhang filled in and T-tailed with Taq polymerase. The remainder of the original pPZP221 multi-cloning site was removed by digestion with EcoRI, and the expression cassette cloned into the EcoRI site and the 3′ T overhang restoring the HindIII site. This binary vector contains between the left and right border the plant selectable marker gene aaaC1 under the control of the 35S promoter and 35S terminator and the pKYLX71:35S2-derived expression cassette with a CaMV 35S promoter with a duplicated enhancer region and an rbcS terminator.
The orientation of the constructs (sense or antisense) was checked by restriction enzyme digest. Transformation vectors containing chimeric genes using full-length open reading frame cDNAs encoding perennial ryegrass F3OH and white clover BANa, CHIa, CHId, CHRc, CHSa1, CHSa3, CHSc, CHSd2, CHSf, CHSh, DFRd, F3Ha, PALa, PALb, PALf and VRa proteins in sense and antisense orientations under the control of the CaMV 35S2 promoter were generated (FIGS. 114, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190 and 195).
Example 6
Production and Analysis of Transgenic Arabidopsis Plants Carrying Chimeric Perennial Ryegrass F3OH and White Clover BANa, CHIa, CHId, CHRc, CHSa1, CHSa3, CHSc, CHSd2, CHSf, CHSh, DFRd, F3Ha, PALa, PALb, PALf and VRa Genes Involved in Flavonoid Biosynthesis
A set of transgenic Arabidopsis plants carrying chimeric perennial ryegrass and white clover genes involved in flavonoid biosynthesis, protein binding, metal chelation, anti-oxidation, UV-light absorption, tolerance to biotic stresses such as viruses, micro-organisms, insects and fungal pathogens; pigmentation in for example flowers and leaves; herbage quality and bloat-safety and isoflavonoid content leading to health benefits, were produced.
pPZP221-based transformation vectors with LpF3OH and TrBANa, TrCHIa, TrCHId, TrCHRc, TrCHSa1, TrCHSa3, TrCHSc, TrCHSd2, TrCHSf, TrCHSh, TrDFRd, TrF3Ha, TrPALa, TrPALb, TrPALf and TrVRa cDNAs comprising the full open reading frame sequences in sense and antisense orientations under the control of the CaMV 35S promoter with duplicated enhancer region (35S2) were generated as detailed in Example 6.
Agrobacterium-mediated gene transfer experiments were performed using these transformation vectors.
The production of transgenic Arabidopsis plants carrying the perennial ryegrass F3OH and white clover BANa, CHIa, CHId, CHRc, CHSa1, CHSa3, CHSc, CHSd2, CHSf, CHSh, DFRd, F3Ha, PALa, PALb, PALf and VRa cDNAs under the control of the CaMV 35S promoter with duplicated enhancer region (35S2) is described here in detail.
Preparation of Arabidopsis Plants
Seedling punnets were filled with Debco seed raising mixture (Debco Pty. Ltd.) to form a mound. The mound was covered with two layers of anti-bird netting secured with rubber bands on each side. The soil was saturated with water and enough seeds (Arabidopsis thaliana ecotype Columbia, Lehle Seeds #WT-02) sown to obtain approximately 15 plants per punnet. The seeds were then vernalised by placing the punnets at 4° C. After 48 hours the punnets were transferred to a growth room at 22° C. under fluorescent light (constant illumination, 55 μmolm-2s-1) and fed with Miracle-Gro (Scotts Australia Pty. Ltd.) once a week. Primary bolts were removed as soon as they appeared. After 4-6 days the secondary bolts were approximately 6 cm tall, and the plants were ready for vacuum infiltration.
Preparation of Agrobacterium 
Agrobacterium tumefaciens strain AGL-1 were streaked on LB medium containing 50 μg/ml rifampicin and 50 μg/ml kanamycin and grown at 27° C. for 48 hours. A single colony was used to inoculate 5 ml of LB medium containing 50 μg/ml rifampicin and 50 μg/ml kanamycin and grown over night at 27° C. and 250 rpm on an orbital shaker. The overnight culture was used as an inoculum for 500 ml of LB medium containing 50 μg/ml kanamycin only. Incubation was over night at 27° C. and 250 rpm on an orbital shaker in a 2 l Erlenmeyer flask.
The overnight cultures were centrifuged for 15 min at 5500×g and the supernatant discarded. The cells were resuspended in 1 l of infiltration medium [5% (w/v) sucrose, 0.03% (v/v) Silwet-L77 (Vac-In-Stuff, Lehle Seeds #VIS-01)] and immediately used for infiltration.
Vacuum Infiltration
The Agrobacterium suspension was poured into a container (Décor Tellfresh storer, #024) and the container placed inside the vacuum desiccator (Bel Art, #42020-0000). A punnet with Arabidopsis plants was inverted and dipped into the Agrobacterium suspension and a gentle vacuum (250 mm Hg) was applied for 2 min. After infiltration, the plants were returned to the growth room where they were kept away from direct light overnight. The next day the plants were returned to full direct light and allowed to grow until the siliques were fully developed. The plants were then allowed to dry out, the seed collected from the siliques and either stored at room temperature in a dry container or used for selection of transformants.
Selection of Transformants
Prior to plating the seeds were sterilized as follows. Sufficient seeds for one 150 mm petri dish (approximately 40 mg or 2000 seeds) were placed in a 1.5 ml microfuge tube. Five hundred microliters (500 μl) 70% ethanol were added for 2 min and replaced by 500 μl sterilization solution (H2O: 4% chlorine: 5% SDS, 15:8:1). After vigorous shaking, the tube was left for 10 min after which time the sterilization solution was replaced with 500 μl sterile water. The tube was shaken and spun for 5 sec to sediment the seeds. The washing step was repeated 3 times and the seeds were left covered with approximately 200 μl sterile water.
The seeds were then evenly spread on 150 mm petri dishes containing germination medium (4.61 g Murashige & Skoog salts, 10 g sucrose, 1 ml 1 M KOH, 2 g Phytagel, 0.5 g MES and 1 ml 1000× Gamborg's B-5 vitamins per litre) supplemented with 250 μg/ml timetin and 75 μg/ml gentamycin. After vernalisation for 48 hours at 4° C. the plants were grown under continuous fluorescent light (55 μmol m-2s-1) at 22° C. to the 6-8 leaf stage and transferred to soil.
Preparation of Genomic DNA
Three to four leaves of Arabidopsis plants regenerated on selective medium were harvested and freeze-dried. The tissue was homogenized on a Retsch MM300 mixer mill, then centrifuged for 10 min at 1700×g to collect cell debris. Genomic DNA was isolated from the supernatant using Wizard Magnetic 96 DNA Plant System kits (Promega) on a Biomek FX (Beckman Coulter). 5 μl of the sample (50 μl) were then analyzed on an agarose gel to check the yield and the quality of the genomic DNA.
Analysis of DNA Using Real-Time PCR
Genomic DNA was analyzed for the presence of the transgene by real-time PCR using SYBR Green chemistry. PCR primer pairs (Table 5) were designed using MacVector (Accelrys). The forward primer was located within the 35S2 promoter region and the reverse primer within the transgene to amplify products of approximately 150-250 bp as recommended. The positioning of the forward primer within the 35S2 promoter region guaranteed that homologous genes in Arabidopsis were not detected.
5 μl of each genomic DNA sample was run in a 50 μl PCR reaction including SYBR Green on an ABI (Applied Biosystems) together with samples containing DNA isolated from wild type Arabidopsis plants (negative control), samples containing buffer instead of DNA (buffer control) and samples containing the plasmid used for transformation (positive plasmid control).
Plants were obtained after transformation with all chimeric constructs and selection on medium containing gentamycin. The selection process and two representative real-time PCR analyses are shown in FIG. 196.








TABLE 5







LIST OF PRIMERS USED FOR REAL-TIME PCR ANALYSIS OF ARABIDOPSIS



PLANTS TRANSFORMED WITH CHIMERIC PERENNIAL RYEGRASS GENES


INVOLVED IN FLAVONOID BIOSYNTHESIS


 














SEQ ID

SEQ ID



CONSTRUCT
PRIMER 1 (FORWARD)
NO
PRIMER 2 (REVERSE)
NO















pPZP221LpF3OH
TTGGAGAGGACACGCTGAAATC
410
AGGAGAGGGTTGGACATCGC
411



sense


 


pPZP221LpF3OH
CATTTCATTTGGAGAGGACACGC
412
ACGAGGAGTTCTGGAAGATGGG
413


anti


 


pPZP221TrBANa
TTGGAGAGGACACGCTGAAATC
414
GCAACAAAACCAGTGCCACC
415


sense


 


pPZP221TrBANa
TCATTTGGAGAGGACACGCTG
416
GATGATTGCCCCAGCAAGG
417


anti


 


pPZP221TrCHIa
CATTTCATTTGGAGAGGACACGC
418
CAAGGTTCTCGACTTGGATTGC
419


sense


 


pPZP221TrCHIa
TCATTTGGAGAGGACACGCTG
420
AGATTACCTGCCTTGTTGAACGAG
421


anti


 


pPZP221TrCHId
TCATTTGGAGAGGACACGCTG
422
GACGGTAGGAGGGAATAGATTGTTC
423


sense


 


pPZP221TrCHId
TCATTTGGAGAGGACACGCTG
424
CCAGGTTATCCGAGTTATTCAACG
425


anti


 


pPZP221TrCHRc
CCACTATCCTTCGCAAGACCC
426
TCCCATTCCAACCACAGGC
427


sense


 


pPZP221TrCHRc
TCATTTGGAGAGGACACGCTG
428
CAAGCCAGGACTCAGTGACCTATG
429


anti


 


pPZP221TrCHSa1
TCATTTGGAGAGGACACGCTG
430
CTGGTCAACACGATTTGCTGG
431


sense


 


pPZP221TrCHSa1
TCATTTGGAGAGGACACGCTG
432
AACCACAGGAGAAGGACTTGACTG
433


anti


 


pPZP221TrCHSa3
CATTTCATTTGGAGAGGACACGC
434
AACACGGTTTGGTGGATTTGC
435


sense


 


pPZP221TrCHSa3
TCATTTGGAGAGGACACGCTG
436
ACAACTGGAGAAGGACTTGATTGG
437


anti


 


pPZP221TrCHSc
TTGGAGAGGACACGCTGAAATC
438
ACAAGTTGGTGAGGGAATGCC
439


sense


 


pPZP221TrCHSc
TCATTTGGAGAGGACACGCTG
440
GGGATTGATACTTGCTTTTGGACC
441


anti


 


pPZP221TrCHSd
CCCACTATCCTTCGCAAGACC
442
AGTTGCAGTGCCGATTGCC
443


2 sense


 


pPZP221TrCHSd
CATTTCATTTGGAGAGGACACGC
444
AAGATGGACTTGCCACAACAGG
445


2 anti


 


pPZP221TrCHSf
CATTTCATTTGGAGAGGACACGC
446
TCGTTGCCTTTCCCTGAGTAGG
447


sense


 


pPZP221TrCHSf
TCATTTGGAGAGGACACGCTG
448
GATTGGCTTTTGGACCAGGG
449


anti


 


pPZP221TrCHSh
TCATTTGGAGAGGACACGCTG
450
CGGTCACCATTTTTTTGTTGGAGG
451


sense


 


pPZP221TrCHSh
TCATTTGGAGAGGACACGCTG
452
TGTTGTTTGGGTTTGGACCG
453


anti


 


pPZP221TrDFRd
CATTTCATTTGGAGAGGACACGC
454
ATTGAGATTTTGGACGGTGGC
455


sense


 


pPZP221TrDFRd
CATTTCATTTGGAGAGGACACGC
456
CGCAACCTGGATTGTTGAGAGC
457


anti


 


pPZP221TrF3Ha
TCATTTGGAGAGGACACGCTG
458
TCTTCCCTAACGAAACTTGACTCG
459


sense


 


pPZP221TrF3Ha
TCATTTGGAGAGGACACGCTG
460
GAACAACAACTTAGGGACTTGGAGG
461


anti


 


pPZP221TrPALa
ATGACGCACAATCCCACTATCC
462
TTGCCTCAGCAGCCACACC
463


sense


 


pPZP221TrPALa
GGAGAGGACACGCTGAAATCAC
464
TGCCAAAAGAGGTTGAAAGTGC
465


anti


 


pPZP221TrPALb
ATCCCACTATCCTTCGCAAGACCC
466
AATGACTCCCCATCAACGACTCCG
467


sense


 


pPZP221TrPALb
TTGGAGAGGACACGCTGAAATC
468
GACAAATTGTTCACAGCTATGTGCC
469


anti


 


pPZP221TrPALf
ATCCCACTATCCTTCGCAAGACCC
470
CACCATACGCTTCACCTCATCC
471


sense


 


pPZP221TrPALf
TCATTTGGAGAGGACACGCTG
472
TTGTTAGAGAGGAGTTAGGAACCGC
473


anti


 


pPZP221TrVRa
CCACTATCCTTCGCAAGACCC
474
GCTTACATCCCTCTTACGTTCTGG
475


sense


 


pPZP221TrVRa
CCACTATCCTTCGCAAGACCC
476
AAAAGCTCGTGGACGCTGG
477


anti









Example 7
Genetic Mapping of Perennial Ryegrass Genes Involved in Flavonoid Biosynthesis, Protein Binding, Metal Chelation, Anti-Oxidation, UV-Light Absorption, Tolerance to Biotic Stresses Such As Viruses, Micro-Organisms, Insects and Fungal Pathogens; Pigmentation in for Example Flowers and Leaves; Herbage Quality and Bloat-Safety and Isoflavonoid Content Leading to Health Benefits
The cDNAs representing genes involved in flavonoid biosynthesis, protein binding, metal chelation, anti-oxidation, UV-light absorption, tolerance to biotic stresses such as viruses, micro-organisms, insects and fungal pathogens; pigmentation in for example flowers and leaves; herbage quality and bloat-safety and isoflavonoid content leading to health benefits, were amplified by PCR from their respective plasmids, gel-purified and radio-labelled for use as probes to detect restriction fragment length polymorphisms (RFLPs). RFLPs were mapped in the F1 (first generation) population, NA6×AU6. This population was made by crossing an individual (NA6) from a North African ecotype with an individual (AU6) from the cultivar Aurora, which is derived from a Swiss ecotype. Genomic DNA of the 2 parents and 114 progeny was extracted using the 1×CTAB method of Fulton et al. (1995).
Probes were screened for their ability to detect polymorphism using the DNA (10 μg) of both parents and 5 F1 progeny restricted with the enzymes DraI, EcoRI, EcoRV or HindIII. Hybridizations were carried out using the method of Sharp et al. (1988). Polymorphic probes were screened on a progeny set of 114 individuals restricted with the appropriate enzyme (FIG. 115).
RFLP bands segregating within the population were scored and the data were entered into an Excel spreadsheet. Alleles showing the expected 1:1 ratio were mapped using MAPMAKER 3.0 (Lander et al. 1987). Alleles segregating from, and unique to, each parent, were mapped separately to give two different linkage maps. Markers were grouped into linkage groups at a LOD of 5.0 and ordered within each linkage group using a LOD threshold of 2.0.
Loci representing genes involved in flavonoid biosynthesis mapped to the linkage groups as indicated in Table 6 and in FIG. 197. These gene locations can now be used as candidate genes for quantitative trait loci associated with flavonoid biosynthesis, protein binding, metal chelation, anti-oxidation, UV-light absorption, tolerance to biotic stresses such as viruses, micro-organisms, insects and fungal pathogens; pigmentation in for example flowers and leaves; herbage quality and bloat-safety and isoflavonoid content leading to health benefits.







TABLE 6







MAP LOCATIONS OF RYEGRASS GENES INVOLVED IN


FLAVONOID BIOSYNTHESIS ACROSS TWO GENETIC


LINKAGE MAPS OF PERENNIAL RYEGRASS















Linkage group












Probe
Polymorphic
Mapped with
Locus
NA6 →
AU6





LpDFRb
Y
Hind III
LpDFRb
6
6









Finally, it is to be understood that various alterations, modifications and/or additions may be made without departing from the spirit of the present invention as outlined herein.
It will also be understood that the term “comprises” (or its grammatical variants) as used in this specification is equivalent to the term “includes” and should not be taken as excluding the presence of other elements or features.
Documents cited in this specification are for reference purposes only and their inclusion is not acknowledgment that they form part of the common general knowledge in the relevant art.
REFERENCES


    An, G., Watson, B. D., Stachel, S., Gordon, M. P., Nester, E. W. (1985) “New cloning vehicles for transformation of higher plants”. The EMBO Journal 4, 227-284.
    Feinberg, A. P., Vogelstein, B. (1984). “A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity”. Anal. Biochem. 132: 6-13.
    Frohman et al. (1988) “Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer”. Proc. Natl. Acad Sci. USA 85:8998.
    Gish and States (1993) “Identification of protein coding regions by database similarity search”. Nature Genetics 3:266-272.
    Lander, E. S., Green P., Abrahamson, J., Barlow, A., Daly, M. J., Lincoln, S. E., Newburg, L. (1987). “MAPMAKER: an interactive computer package for constructing primary linkage maps of experimental and natural populations”. Genomics 1: 174-181.
    Loh, E. Y., Elliott, J. F., Cwirla, S., Lanier, L. L., Davis, M. M. (1989). “Polymerase chain reaction with single-sided specificity: Analysis of T-cell receptor delta chain”. Science 243:217-220.
    Ohara, O., Dorit, R. L., Gilbert, W. (1989). “One-sided polymerase chain reaction: The amplification of cDNA”. Proc. Natl. Acad Sci USA 86:5673-5677.
    Sambrook, J., Fritsch, E. F., Maniatis, T. (1989). Molecular Cloning, A Laboratory Manual. Cold Spring Harbour Laboratory Press.
    Schardl, C. L., Byrd, A. D., Benzion, G., Altschuler, M. A., Hildebrand, D. F., Hunt, A. G. (1987) “Design and construction of a versatile system for the expression of foreign genes in plants”. Gene 61, 1-11.
    Sharp, P. J., Kreis, M., Shewry, P. R., Gale, M. D. (1988). “Location of α-amylase sequences in Wheat and its relatives”. Theor. Appl. Genet. 75:286-290.

## Claims

1. A substantially purified or isolated nucleic acid comprising a nucleotide sequence selected from the group consisting of:
(a) the nucleotide sequence of SEQ ID NO:305;
(b) the nucleotide sequence comprising the coding portion of SEQ ID NO: 305;
(c) the nucleotide sequence which is the full length complement of SEQ ID NO: 305; and
(d) the nucleotide sequence which is the full length complement of the coding portion of SEQ ID NO: 305.

2. A construct including the nucleic acid according to claim 1.

3. A vector including the nucleic acid or nucleic acid according to claim 1.

4. The vector according to claim 3, further including a promoter and a terminator, said promoter, nucleic acid and terminator being operatively linked.

5. A plant cell, plant, plant seed or other plant part, having incorporated therein the vector according to claim 3.

6. A plant, plant seed or other plant part derived from the plant cell or plant according to claim 5 and having incorporated therein the vector according to claim 3.

7. A method selected from the group consisting of:
(a) modifying flavonoid biosynthesis in a plant;
(b) modifying protein binding, metal chelation, anti-oxidation, and/or UV-light absorption in a plant;
(c) modifying pigment production in plant;
(d) modifying plant defense to a biotic stress; and
(e) modifying forage quality of a plant by disrupting protein foam and/or conferring protection from rumen pasture bloat;
said method including introducing into said plant an effective amount of a nucleic acid according to claim 1.

8. A method according to claim 7 wherein said biotic stress is selected from the group consisting of viruses, microorganisms, insects and fungal pathogens.

9. A substantially purified or isolated nucleic acid encoding BAN, said nucleic acid comprising the nucleotide sequence of SEQ ID NO: 305.

10. A substantially purified or isolated nucleic acid comprising a nucleotide sequence selected from the group consisting of:
(a) the nucleotide sequence of SEQ ID NO:305;
(b) the nucleotide sequence comprising the coding portion of SEQ ID NO: 305;
(c) the nucleotide sequence which is the full length complement of SEQ ID NO: 305;
(d) the nucleotide which is the full length complement of the coding portion of Seq ID No. 305;
(e) a nucleotide sequence which is a variant of SEQ ID NO:305 with one or more nucleotide changes that result in conservative amino acid substitutions in the encoded polypeptide with the proviso that the variant has at least approximately 98% identity to SEQ ID NO: 305; and
(f) a nucleotide sequence which is a variant of the coding portion of SEQ ID NO: 305 with one or more nucleotide changes that result in conservative amino acid substitutions in the encoded polypeptide with the proviso that the variant has at least approximately 98% identity to the coding portion of SEQ ID NO: 305.

11. A substantially purified or isolated nucleic acid encoding BAN, said nucleic acid or nucleic acid fragment comprising the nucleotide sequence that is a variant of SEQ ID NO: 305 with one or more nucleotide changes that result in conservative amino acid substitutions in the encoded polypeptide with the proviso that the variant has at least approximately 98% identity to SEQ ID No.: 305.