# Patent

## Title
Chalcone synthase dihyrdoflavonol 4-reductase and leucoanthocyanidine reductase from clover, medic ryegrass or fescue

## Abstract

The present invention relates to nucleic acid fragments encoding amino acid sequences for flavonoid biosynthetic enzymes in plants, and the use thereof for the modification of, for example, flavonoid biosynthesis in plants, and more specifically the modification of the content of condensed tannins. In particularly preferred embodiments, the invention relates to the combinatorial expression of chalcone synthase (CHS) and/or dihydroflavonol 4-reductase (BAN) and/or leucoanthocyanidine reductase (LAR) in plants to modify, for example, flavonoid biosynthesis or more specifically the content of condensed tannins.

## Background
The present invention relates to nucleic acid fragments encoding amino acid sequences for flavonoid biosynthetic enzyme polypeptides in plants, and the use thereof for the modification of, for example, flavonoid biosynthesis in plants, and more specifically the modification of the content of condensed tannins. In particularly preferred embodiments, the invention relates to the combinatorial expression of chalcone synthase (CHS) and/or dihydroflavonol 4-reductase (BAN) and/or leucoanthocyanidine reductase (LAR) in plants to modify, for example, flavonoid biosynthesis or more specifically the content of condensed tannins.
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 specialised 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 signalling between plants and microbes, in male fertility of some species, in defence 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. Condensed tannins which are plant polyphenols with protein-precipitating and antioxidant properties are involved in protein binding, metal chelation, anti-oxidation, and UV-light absorption. As a result condensed tannins inhibit viruses, microorganisms, 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.
The major branch pathways of flavonoid biosynthesis start with general phenylpropanoid metabolism and lead to the nine major subgroups: the colourless 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 channeling carbon into the flavonoid biosynthesis pathway is catalysed by chalcone synthase (CHS), which uses malonyl CoA and 4-coumaryl CoA as substrates.
The Arabidopsis BANYULS gene encodes a dihydroflavonol 4-reductase-like protein (BAN) that may be an anthocyanine reductase (ACR). The reaction catalysed by BAN is considered to be one possible branching point from the general flavonoid pathway to the condensed tannin biosynthesis.
An alternative pathway to condensed tannins is via leucoanthocyanidine reductase (LAR). LAR utilises the same substrate as the ACR (BAN) but produces a 2,3-trans isomer as compared to the 2,3-cis isomer produced by ACR.
While nucleic acid sequences encoding the key enzymes in the condensed tannins biosynthetic pathway CHS, BAN and LAR have been isolated for certain species of plants, there remains a need for materials useful in modifying flavonoid biosynthesis and more specifically in modifying condensed tannin biosynthesis and therewith in modifying forage quality, for example by disrupting protein foam and 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.
In one aspect, the present invention provides substantially purified or isolated nucleic acids or nucleic acid fragments encoding key polypeptide enzymes in the condensed tannins biosynthetic pathway CHS, BAN and LAR, or functionally active fragments or variants of these enzymes, from a clover (Trifolium), medic (Medicago), ryegrass (Lolium) or fescue (Festuca) species.
The present invention also provides substantially purified or isolated nucleic acids or nucleic acid fragments encoding amino acid sequences for a class of polypeptides which are related to CHS, BAN and LAR or functionally active fragments or variants of CHS, BAN or LAR. Such polypeptides are referred to herein as CHS-like, BAN-like and LAR-like, respectively, and includes polypeptides having similar functional activity.
The individual or simultaneous enhancement or otherwise manipulation of CHS, BAN and LAR 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, and UV-light absorption; may enhance or reduce or otherwise alter plant pigment production; and may enhance or otherwise alter the amount of condensed tannins contained within forage legumes and grasses, including alfalfa, medics, clovers, ryegrasses and fescues and therewith the capacity to reduce bloating by disrupting protein foam.
Methods for the manipulation of CHS, BAN and LAR 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, microorganisms, insects and fungal pathogens; altered pigmentation in flowers; forage legumes with enhanced herbage quality and bloat-safety.
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 RNA is readily obtainable, for example, by transcription of a DNA sequence according to the present invention, to produce an RNA corresponding to the DNA sequence. The RNA may be synthesised, in vivo or in vitro or by chemical synthesis to produce a sequence corresponding to a DNA sequence by methods well known in the art. In this specification, where the degree of sequence similarity between an RNA and DNA is such that the strand of the DNA could encode the RNA, then the RNA is said to “correspond” to that DNA.
In a 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 the nucleotide sequences shown in FIGS. 2, 6, 10 and 14 hereto (Sequence ID Nos. 1, 3, 5 and 7, 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); and (e) RNA sequences corresponding to the sequences recited in (a), (b), (c), and (d).
In a further preferred embodiment of this aspect of the invention, the substantially purified or isolated nucleic acid or nucleic acid fragment encoding a BAN or BAN-like protein includes the nucleotide sequence shown in FIG. 18 hereto (Sequence ID No. 9); (b) complements of the sequence 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); and (e) RNA sequences corresponding to the sequences recited in (a), (b), (c), and (d).
In a still further preferred embodiment of this aspect of the invention, the substantially purified or isolated nucleic acid or nucleic acid fragment encoding a LAR or LAR-like protein includes the nucleotide sequence shown in FIGS. 22, 26 and 30 hereto (Sequence ID Nos. 11, 13 and 15 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); and (e) RNA sequences corresponding to the sequences recited in (a), (b), (c), and (d).
The term “isolated” means that the material is removed from its original environment (e.g. the natural environment if it is naturally occurring). For example, a naturally occurring nucleic acid or polypeptide present in a living plant is not isolated, but the same nucleic acid or polypeptide 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. An isolated polypeptide could be part of a composition and still be isolated in that such a composition is not part of its natural environment.
The term “purified” means that the nucleic acid or polypeptide is substantially free of other nucleic acids or polypeptides.
By “functionally active” in respect of a nucleic acid it is meant that the fragment or variant (such as an analogue, derivative or mutant) 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 sequence, more preferably at least approximately 90% identity, most preferably at least approximately 95% identity. 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 30 nucleotides, more preferably at least 45 nucleotides, most preferably at least 60 nucleotides.
By “functionally active” in respect of a polypeptide is meant that the fragment or variant has one or more of the biological properties or functions of the polypeptides CHS, CHS-like, BAN, BAN-like, LAR and LAR-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 sequence, more preferably at least approximately 80% identity, most preferably at least approximately 90% identity. 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.
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.
The term “vector” as used herein encompasses both cloning and expression vectors. Vectors are often recombinant molecules containing nucleic acid molecules from several sources.
By “operatively linked” is meant that said regulatory element(s) is capable of causing expression of said nucleic acid(s) or nucleic acid fragment(s) in a plant cell and said terminator(s) is capable of terminating expression of said nucleic acid(s) or nucleic acid fragment(s) in a plant cell. Preferably, said regulatory element(s) is upstream of said nucleic acid(s) or nucleic acid fragment(s) and said terminator(s) is downstream of said nucleic acid(s) or nucleic acid fragment(s). In a particularly preferred embodiment, each nucleic acid or nucleic acid fragment has one or more upstream promoters and one or more downstream terminators, although expression of more than one nucleic acid or nucleic acid fragment from an upstream regulatory element(s) or termination of more than one nucleic acid or nucleic acid fragment from a downstream terminator(s) is not precluded.
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, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, the entire disclosure of which is incorporated herein by reference.
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.
Genes encoding other CHS or CHS-like, BAN or BAN-like and LAR or LAR-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 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 CHS and CHS-like, BAN and BAN-like, and LAR and LAR-like proteins; 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 CHS or CHS-like polypeptide includes an amino acid sequence selected from the group consisting of sequences shown in FIGS. 3, 7, 11 and 15 hereto (Sequence ID Nos. 2, 4, 6 and 8, respectively) and functionally active fragments and variants thereof.
In a further preferred embodiment of this aspect of the invention, the substantially purified or isolated BAN or BAN-like polypeptide includes an amino acid sequence shown in FIG. 19 hereto (Sequence ID No. 10), and functionally active fragments and variants thereof.
In a still further preferred embodiment of this aspect of the invention, the substantially purified or isolated LAR or LAR-like polypeptide includes an amino acid sequence selected from the group consisting of sequences shown in FIGS. 23, 27 and 31 hereto (Sequence ID Nos. 12, 14 and 16, respectively), and functionally active fragments and variants thereof.
In a further embodiment of this aspect of the invention, there is provided a polypeptide produced (e.g. recombinantly) 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.
In a still further aspect of the present invention there is provided a construct including one or more nucleic acids or nucleic acid fragments according to the present invention.
In a particularly preferred embodiment the construct may include nucleic acids or nucleic acid fragments encoding both CHS or CHS-like and BAN or BAN-like polypeptides.
In another preferred embodiment the construct may include nucleic acids or nucleic acid fragments encoding both CHS or CHS-like and LAR or LAR-like polypeptides.
In yet another preferred embodiment the construct may include nucleic acids or nucleic acid fragments encoding both LAR or LAR-like and BAN or BAN-like polypeptides.
In an even more preferred embodiment the construct may include nucleic acids or nucleic acid fragments encoding all three of CHS or CHS-like, BAN or BAN-like and LAR or LAR-like polypeptides.
Constructs including nucleic acids or nucleic acid fragments encoding CHS or CHS-like and BAN or BAN-like, and optionally further including nucleic acids or nucleic acid fragments encoding LAR or LAR-like, are particularly preferred.
In a still further aspect of the present invention there is provided a vector including one or more nucleic acids or nucleic acid fragments according to the present invention.
In a preferred embodiment of this aspect of the invention, the construct may include one or several of the following: one or more regulatory elements such as promoters, one or more nucleic acids or nucleic acid fragments according to the present invention and one or more terminators; said on or more regulatory elements, one or more nucleic acids or nucleic acid fragments and one or more terminators being operatively linked.
In a particularly preferred embodiment the construct may contain nucleic acids or nucleic acid fragments encoding both CHS or CHS-like and BAN or BAN-like polypeptides, operatively linked to a regulatory element or regulatory elements, such that both CHS or CHS-like and BAN or BAN-like polypeptides are expressed.
In another preferred embodiment the construct may contain nucleic acids or nucleic acid fragments encoding both CHS or CHS-like and LAR or LAR-like polypeptides, operatively linked to a regulatory element or regulatory elements, such that both CHS or CHS-like and LAR or LAR-like polypeptides are expressed.
In yet another preferred embodiment the construct may contain nucleic acids or nucleic acid fragments encoding both LAR or LAR-like and BAN or BAN-like polypeptides, operatively linked to a regulatory element or regulatory elements, such that both LAR or LAR-like and BAN or BAN-like polypeptides are expressed.
In an even more preferred embodiment the construct may contain nucleic acids or nucleic acid fragments encoding all three of CHS or CHS-like, BAN or BAN-like and LAR or LAR-like polypeptides, operatively linked to a regulatory element or regulatory elements, such that all three of CHS or CHS-like, BAN or BAN-like and LAR or LAR-like polypeptides are expressed.
Constructs including nucleic acids or nucleic acid fragments encoding CHS or CHS-like and BAN or BAN-like, and optionally further including nucleic acids or nucleic acid fragments encoding LAR or LAR-like, are particularly preferred.
The construct or 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 promoters include but are not limited to the constitutive Cauliflower Mosaic Virus 35S (CaMV 35S) promoter and derivatives thereof, the maize Ubiquitin promoter, the rice Actin promoter, and the tissue-specific Arabidopsis small subunit (ASSU) promoter.
A variety of terminators which may be employed in the vectors and constructs 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 construct or vector, in addition to the regulatory element(s), the nucleic acid(s) or nucleic acid fragment(s) of the present invention and the terminator(s), may include further elements necessary for expression of the nucleic acid(s) or nucleic acid fragment(s), in different combinations, for example vector backbone, origin of replication (ori), multiple cloning sites, recognition sites for recombination events, 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, visual examination including microscopic examination of fluorescence emitted by gfp, northern and Western blot hybridisation analyses.
Those skilled in the art will appreciate that the various components of the construct or vector are operatively linked, so as to result in expression of said nucleic acid(s) or nucleic acid fragment(s). 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 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 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.
In a further aspect of the present invention there is provided a method of isogenic transformation of a dicotyledonous plant, said method including transforming only one of each pair of cotyledons. This enables the production of pairs of transgenic plant and corresponding untransformed negative control in an otherwise isogenic genetic background for detailed functional assessment of the impact of the transgene on plant phenotype. In a preferred embodiment of this aspect of the invention, the method may include isogenic transformation of a dicotyledonous plant with a construct or vector according to the present invention.
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, one or more constructs, vectors, nucleic acids or nucleic acid fragments 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), 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 condensed tannin biosynthesis; of modifying flavonoid biosynthesis; of modifying protein binding, metal chelation, anti-oxidation, and UV-light absorption; of modifying plant pigment production; of modifying plant defence to biotic stresses such as viruses, microorganisms, insects, fungal pathogens; of modifying forage quality by disrupting protein foam and 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 vector according to the present invention.
In a particularly preferred embodiment the method may include introducing into said plant nucleic acids or nucleic acid fragments encoding both CHS or CHS-like and BAN or BAN-like polypeptides.
In another preferred embodiment the method may include introducing into said plant nucleic acids or nucleic acid fragments encoding both CHS or CHS-like and LAR or LAR-like polypeptides.
In yet another preferred embodiment the method may include introducing into said plant nucleic acids or nucleic acid fragments encoding both LAR or LAR-like and BAN or BAN-like polypeptides.
In an even more preferred embodiment the method may include introducing into said plant nucleic acids or nucleic acid fragments encoding all three of CHS or CHS-like, BAN or BAN-like and LAR or LAR-like polypeptides.
Methods including the combinatorial expression of nucleic acids or nucleic acid fragments encoding CHS or CHS-like and BAN or BAN-like, and optionally further including the use of nucleic acids or nucleic acid fragments encoding LAR or LAR-like, are particularly preferred.
In a further aspect of the present invention there is provided a method of inhibiting bloat in an animal, said method including providing the animal with a forage plant including a construct, vector, nucleic acid or nucleic acid fragment according to the present invention. The animal is preferably a ruminant, including sheep, goats and cattle. The forage plant including a construct vector, nucleic acid or nucleic acid fragment according to the present invention may form all or part of the feed of the animal. The forage plant preferably expresses CHS or CHS-like proteins, BAN or BAN-like proteins, and/or LAR or LAR-like proteins at higher levels than the equivalent wild-type plant. More preferably, the forage plant expresses both CHS or CHS-like proteins and BAN or BAN-like proteins; both CHS or CHS-like proteins and LAR or LAR-like proteins; or both BAN or BAN-like proteins and LAR or LAR-like proteins; at higher levels than the equivalent wild-type plant. More preferably, the forage plant expresses all three of CHS or CHS-like proteins, BAN or BAN-like proteins, and LAR or LAR-like proteins; at higher levels than the equivalent wild-type plant.
In a further aspect of the present invention there is provided a method for enhancing an animal's growth rate, said method including providing the animal with a forage plant including a construct, vector, nucleic acid or nucleic acid fragment according to the present invention. The animal is preferably a ruminant, including sheep, goats and cattle. The forage plant including a construct, vector, nucleic acid or nucleic acid fragment according to the present invention may form all or part of the feed of the animal. The forage plant preferably expresses CHS or CHS-like proteins, BAN or BAN-like proteins, and/or LAR or LAR-like proteins at higher levels than the equivalent wild-type plant. More preferably, the forage plant expresses both CHS or CHS-like proteins and BAN or BAN-like proteins; both CHS or CHS-like proteins and LAR or LAR-like proteins; or both BAN or BAN-like proteins and LAR or LAR-like proteins; at higher levels than the equivalent wild-type plant. More preferably, the forage plant expresses all three of CHS or CHS-like proteins, BAN or BAN-like proteins, and LAR or LAR-like proteins; at higher levels than the equivalent wild-type plant.
It is estimated that the method of enhancing an animal's growth rate according to this invention should result in an increase in, for example, lamb growth rate of at least approximately 5%, more preferably at least approximately 10%.
Using the methods and materials of the present invention, condensed tannin biosynthesis, flavonoid biosynthesis, protein binding, metal chelation, anti-oxidation, UV-light absorption, tolerance to biotic stresses such as viruses, microorganisms, insects and fungal pathogens; pigmentation in for example flowers and leaves; herbage quality and bloat-safety; isoflavonoid content leading to health benefits, may be increased or otherwise altered, for example by incorporating additional copies of one or more sense nucleic acids or nucleic acid fragments of the present invention. They may be decreased or otherwise altered, for example by incorporating one or more antisense nucleic acids or nucleic acid fragments of the present invention.
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.
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.



In the Figures
FIG. 1 shows the plasmid map in pGEM-T Easy of TrCHSa3.
FIG. 2 shows the nucleotide sequence of TrCHSa3 (Sequence ID No. 1).
FIG. 3 shows the deduced amino acid sequence of TrCHSa3 (Sequence ID No. 2).
FIG. 4 shows plasmid maps of sense and antisense constructs of TrCHSa3 in the binary vector pPZP221:35 S2.
FIG. 5 shows the plasmid map in pGEM-T Easy of TrCHSc.
FIG. 6 shows the nucleotide sequence of TrCHSc (Sequence ID No. 3).
FIG. 7 shows the deduced amino acid sequence of TrCHSc (Sequence ID No. 4).
FIG. 8 shows plasmid maps of sense and antisense constructs of TrCHSc in the binary vector pPZP221:35 S2.
FIG. 9 shows the plasmid map in pGEM-T Easy of TrCHSf.
FIG. 10 shows the nucleotide sequence of TrCHSf (Sequence ID No. 5).
FIG. 11 shows the deduced amino acid sequence of TrCHSf (Sequence ID No. 6).
FIG. 12 shows plasmid maps of sense and antisense constructs of TrCHSf in the binary vector pPZP221:35 S2.
FIG. 13 shows the plasmid map in pGEM-T Easy of TrCHSh.
FIG. 14 shows the nucleotide sequence of TrCHSh (Sequence ID No. 7).
FIG. 15 shows the deduced amino acid sequence of TrCHSh (Sequence ID No. 8).
FIG. 16 shows plasmid maps of sense and antisense constructs of TrCHSh in the binary vector pPZP221:35 S2.
FIG. 17 shows the plasmid map in pGEM-T Easy of TrBANa.
FIG. 18 shows the nucleotide sequence of TrBANa (Sequence ID No. 9).
FIG. 19 shows the deduced amino acid sequence of TrBANa (Sequence ID No. 10).
FIG. 20 shows plasmid maps of sense and antisense constructs TrBANa in the binary vector pPZP221:35 S2.
FIG. 21 shows the plasmid map in pGEM-T Easy of TrLARa.
FIG. 22 shows the nucleotide sequence of TrLARa (Sequence ID No. 11).
FIG. 23 shows the deduced amino acid sequence of TrLARa (Sequence ID No. 12).
FIG. 24 shows plasmid maps of sense and antisense constructs of TrLARa in the binary vector pPZP221:35 S2.
FIG. 25 shows the plasmid map in pGEM-T Easy of TrLARb.
FIG. 26 shows the nucleotide sequence of TrLARb (Sequence ID No. 13).
FIG. 27 shows the deduced amino acid sequence of TrLARb (Sequence ID No. 14).
FIG. 28 shows plasmid maps of sense and antisense constructs of TrLARb in the binary vector pPZP221:35 S2.
FIG. 29 shows the plasmid map in pGEM-T Easy of TrLARc.
FIG. 30 shows the nucleotide sequence of TrLARc (Sequence ID No. 15).
FIG. 31 shows the deduced amino acid sequence of TrLARc (Sequence ID No. 16).
FIG. 32 shows plasmid maps of sense and antisense constructs of TrLARc in the binary vector pPZP221:35 S2.
FIG. 33 shows the plasmid map of the binary vector pPZP221:ASSU::TrBAN:35S2::TrCHS.
FIG. 34 shows the plasmid maps of the modular vector system comprising a binary base vector and 7 auxiliary vectors.
FIG. 35 shows an example of the modular binary transformation vector system comprising plasmid maps of the binary transformation vector backbone and 4 expression cassettes in auxiliary vectors (A) and the plasmid map of the T-DNA region of the final binary transformation vector.
FIG. 36 shows A, white clover cotyledons; B, C, D, selection of plantlets transformed with a binary transformation vector constructed as described in Examples 4 and 5; E, putative transgenic white clover on root-inducing medium; F, G, white clover plants transgenic for genes involved in condensed tannin biosynthesis.
FIG. 37 shows the molecular analysis of white clover plants transgenic for the TrBAN gene with Q-PCR amplification plot, agarose gel of PCR product and Southern hybridisation blot.
FIG. 38 shows the molecular analysis of white clover plants transgenic for the TrCHSf gene with Q-PCR amplification plot and agarose gel of PCR product.
FIG. 39 shows the molecular analysis of white clover plants transgenic for the TrLARb gene with Q-PCR amplification plot, agarose gel of PCR product and Southern hybridisation blot.



EXAMPLE 1
Preparation of cDNA Libraries, Isolation and Sequencing of cDNAs Coding for CHS, CHS-like, BAN, BAN-like, LAR and LAR-Like Proteins from White Clover Trifolium repens 
cDNA libraries representing mRNAs from various organs and tissues of white clover (Trifolium repens) were prepared. The characteristics of the white clover libraries are described below (Table 1).







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









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 Epicurean 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 are prepared from randomly picked bacterial colonies containing recombinant plasmids, or the insert cDNA sequences are amplified via polymerase chain reaction using primers specific for vector sequences flanking the inserted cDNA sequences. Plasmid DNA preparation may be performed robotically using the Qiagen QiaPrep Turbo kit (Qiagen, Germany) according to the protocol provided by Qiagen. Amplified insert DNAs are sequenced in dye-terminator sequencing reactions to generate partial cDNA sequences (expressed sequence tags or “ESTs”). The resulting ESTs are analysed using an Applied Biosystems ABI 3700 sequence analyser.
EXAMPLE 2
DNA Sequence Analyses
The cDNA clones encoding CHS, CHS-like, BAN, BAN-like, LAR and LAR-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 analysed 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 White Clover CHS, BAN and LAR Proteins
To fully characterise for the purposes of the generation of probes for hybridisation experiments and the generation of transformation vectors, a set of cDNAs encoding white clover CHS, BAN and LAR 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. 48108, USA). 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 identity and one of the three clones was picked for full-length sequencing, usually the one with the best initial sequencing result.
Sequencing of TrBAN could be completed with M13F and M13R primers. Sequencing of TrCHSa3, TrCHSc, TrCHSf, TrCHSh, TrLARa, TrLARb and TrLARc was completed by primer walking, i.e. oligonucleotide primers were designed to the initial sequence and used for further sequencing. The sequences of the oligonucleotide primers are shown in Table 2.
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 TrCHSa3, TrCHSc, TrCHSf, TrCHSh, TrBANa, TrLARa, TrLARb and TrLARc proteins were obtained (FIGS. 1, 2, 5, 6, 9, 10, 13, 14, 17, 18, 21, 22, 25, 26, 29 and 30).







TABLE 2







List of primers used for sequencing of the full-length cDNAs of TrCHSa3,


TrCHSc, TrCHSf, TrCHSh, TrLARa, TrLARb and TrLARc










gene name
clone ID
sequencing primer
primer sequence (5′ > 3′)





TrCHSa3
05wc1RsB06
05wc1RsB06.f1
AGGAGGCTGCAGTCAAGG


 




05wc1RsB06.f2
TGCCTGAAATTGAGAAACC


 




05wc1RsB06.f3
AAAGCTAGCCTTGAAGCC


 


TrCHSc
07wc1TsE12
07wc1TsE12.f1
TCGGACATAACTCATGTGG


 




07wc1TsE12.f2
TTGGGTTGGAGAATAAGG


 




07wc1TsE12.r1
TGGACATTTATTGGTTGC


 




07wc1TsE12.r2
TATCATGTCTGGAAATGC


 


TrCHSf
07wc1UsD07
07wc1UsD07.f1
AGATTGCATCAAAGAATGG


 




07wc1UsD07.r1
GGTCCAAAAGCCAATCC


 


TrCHSh
13wc2lsG04
13wc2lsG04.f1
TAAGACGAGACATAGTGG


 




13wc2lsG04.r1
TATTCACTAAGCACATGC


 


TrLARa
05wc1CsA02
05wc1CsA02.f1
TCATTTCTGCAATAGGAGG


 




05wc1CsA02.r1
ATCCACCTCAGGTGAACC


 


TrLARb
05wc3EsA03
05wc3EsA03.f1
AATAGGAGGCTCTGATGG


 




05wc3EsA03r1
ATCCACCTCAGGTGAACC


 


TrLARc
07wc1VsF06
07wc1VsF06.f1
AGGCTCTGATGGCTTGC


 




07wc1VsF06.r1
ATCCACCTCAGGTGAACC









EXAMPLE 4
Development of Binary Transformation Vectors Containing Chimeric Genes with cDNA Sequences from White Clover TrCHSa3, TrCHSc, TrCHSf, TrCHSh, TrBANa, TrLARa, TrLARb and TrLARc
To alter the expression of the proteins involved in flavonoid biosynthesis, and more specifically condensed tannin biosynthesis to improve herbage quality and bloat-safety, a set of sense and antisense binary transformation vectors was produced.
cDNA fragments were generated by high fidelity PCR with a proofreading DNA polymerase using the original pGEM-T Easy plasmid cDNA as a template. The primers used (Table 3) 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 pKYLX71:35 S2 (Schardl et al., 1987) as follows: pKYLX71:35 S2 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 aacC1 under the control of the 35S promoter and 35S terminator and the pKYLX71:35 S2-derived expression cassette with a CaMV 35S promoter with a duplicated enhancer region and an rbcS terminator.
Alternatively, the primers for the amplification of cDNA fragments contained attB sequences for use with recombinases utilising the GATEWAY® system (Invitrogen). The resulting PCR fragments were used in a recombination reaction with pDONR® vector (Invitrogen) to generate entry vectors. A GATEWAY® cloning cassette (Invitrogen) was introduced into the multicloning site of the pPZP221:35 S2 vector following the manufacturer's protocol. In a further recombination reaction, the cDNAs encoding the open reading frame sequences were transferred from the entry vector to the GATEWAY®-enabled pPZP221:35 S2 vector.
The orientation of the constructs (sense or antisense) was checked by restriction enzyme digest and sequencing which also confirmed the correctness of the sequence. Transformation vectors containing chimeric genes using full-length open reading frame cDNAs encoding white clover TrCHSa3, TrCHSc, TrCHSf, TrCHSh, TrBANa, TrLARa, TrLARb and TrLARc proteins in sense and antisense orientation under the control of the CaMV 35S2 promoter were generated (FIGS. 4, 8, 12, 16, 20, 24, 28 and 32).







TABLE 3







List of primers used to PCR-amplify


the open reading frames









gene




name
primer
primer sequence (5′ −> 3′)





TrCHSa3
05wc1RsB06f
GAATTCTAGAAGATATGGTGAGTGTA




GCTG


 



05wc1RsB06r
GAATTCTAGAATCACACATCTTATAT




AGCC


 


TrCHSa3
05wc1RsB06fG
GGGGACAAGTTTGTACAAAAAAGCAG




GCTTCTAGAAGATATGGTGAGTGTAG




CTG


 



05wc1RsB06rG
GGGGACCACTTTGTACAAGAAAGCTG




GGTTCTAGAATCACACATCTTATATA




GCC


 


TrCHSc
07wc1TsE12f
GAATTCTAGAAGAAGAAATATGGGAG




ACGAAGG


 



07wc1TsE12r
GAATTCTAGAAAGACTTCATGCACAC




AAGTTCC


 


TrCHSf
07wc1UsD07f
GAATTCTAGATGATTCATTGTTTGTT




TCCATAAC


 



07wc1UsD07r
GAATTCTAGAACATATTCATCTTCCT




ATCAC


 


TrCHSh
13wc21sG04f
GAATTCTAGATCCAAATTCTCGTACC




TCACC


 



13wc21sG04r
GAATTCTAGATAGTTCACATCTCTCG




GCAGG


 


TrBANa
05wc2XsG02f
GGATCCTCTAGAGCACTAGTGTGTAT




AAGTTTCTTGG


 



05wc2XsG02r
GGATCCTCTAGACCCCCTTAGTCTTA




AAATACTCG


 


TrLARa
05wc1CsA02fG
GGGGACAAGTTTGTACAAAAAAGCAG




GCTCTAGAAAGCAAAGCAATGGCACC


 



05wc1CsA02rG
GGGGACCACTTTGTACAAGAAAGCTG




GGTCTAGATCCACCTCAGGTGAACC


 


TrLARb
05wc3EsA03fG
GGGGACAAGTTTGTACAAAAAAGCAG




GCTCTAGAAAGCAATGGCACCAGCAG




C


 



05wc3EsA03rG
GGGGACCACTTTGTACAAGAAAGCTG




GGTCTAGATCCACCTCAGGTGAACC


 


TrLARc
07wc1VsF06fG
GGGGACAAGTTTGTACAAAAAAGCAG




GCTCTAGATAAAGCAATGGCACCAGC


 



07wc1VsF06rG
GGGGACCACTTTGTACAAGAAAGCTG




GGTCTAGATCCACCTCAGGTGAACC









The pPZP221:35 S2 binary vector was further modified to contain two expression cassettes within one T-DNA. The expression cassette from the binary vector pWM5 consisting of the ASSU promoter and the tob terminator was PCR-amplified with a proofreading DNA polymerase using oligonucleotide primers with the following sequences:








forward primer
5′-CCACCATGTTTGAAATTTATTATGTGTTTT



TTTCCG-3′;


 


reverse primer
5′-TAATCCCGGGTAAGGGCAGCCCATACAAAT



GAAGC-3′.






The PCR product was cut with BstXI and SmaI and cloned directionally into the equally cut pPZP221:35 S2 vector. Additionally, a GATEWAY® cloning cassette (Invitrogen) was introduced into the multicloning site in the 35S2:rbcS expression cassette following the manufacturer's protocol. TrBANa was cloned into the ASSU:tob expression cassette, TrCHSa3 was amplified with the GATEWAY®-compatible primers (see Table 3) and cloned into the 35S2:rbcS expression cassette. A transformation vector containing chimeric genes using full-length open reading frame cDNAs encoding white clover TrBANa protein in sense orientation under the control of the ASSU promoter and TrCHSc3 protein in sense orientation under the control of the CaMV 35S2 promoter within the same T-DNA was generated (FIG. 33).
EXAMPLE 5
Development of Binary Transformation Vectors Containing Chimeric Genes with a Combination of 2 or More cDNA Sequences from White Clover TrCHSa3, TrCHSc, TrCHSf, TrCHSh, TrBANa, TrLARa, and TrLARc
To alter the expression of the proteins involved in flavonoid biosynthesis, and more specifically condensed tannin biosynthesis to improve herbage quality and bloat-safety, a modular binary transformation vector system was used (FIG. 34). The modular binary vector system enables simultaneous integration of up to seven transgenes the expression of which is controlled by individual promoter and terminator sequences into the plant genome (Goderis et al., 2002).
The modular binary vector system consists of a pPZP200-derived vector (Hajdukiewicz et al., 1994) backbone containing within the T-DNA a number of unique restriction sites recognised by homing endonucleases. The same restriction sites are present in pUC18-based auxiliary vectors flanking standard multicloning sites. Expression cassettes comprising a selectable marker gene sequence or a cDNA sequence to be introduced into the plant under the control of regulatory sequences like promoter and terminator can be constructed in the auxiliary vectors and then transferred to the binary vector backbone utilising the homing endonuclease restriction sites. Up to seven expression cassettes can thus be integrated into a single binary transformation vector. The system is highly flexible and allows for any combination of cDNA sequence to be introduced into the plant with any regulatory sequence.
For example, a selectable marker cassette comprising the nos promoter and nos terminator regulatory sequences controlling the expression of the nptII gene was PCR-amplified using a proofreading DNA polymerase from the binary vector pKYLX71:35 S2 and directionally cloned into the AgeI and NotI sites of the auxiliary vector pAUX3166. Equally, other selectable marker cassettes can be introduced into any of the auxiliary vectors.
In another example, the expression cassette from the binary vector pWM5 consisting of the ASSU promoter and the tob terminator was PCR-amplified with a proofreading DNA polymerase and directionally cloned into the AgeI and NotI sites of the auxiliary vector pAUX3169. Equally, other expression cassettes can be introduced into any of the auxiliary vectors.
In yet another example, the expression cassette from the direct gene transfer vector pDH51 was cut using EcoRI and cloned directly into the EcoRI site of the auxiliary vector pAUX3132.







TABLE 4







List of primers used to PCR-amplify plant


selectable marker cassettes and the regulatory


elements used to control the expression of


TrCHSa3, TrCHSc, TrCHSf, TrCHSh, TrBANa,


TrLARa, TrLARb and TrLARc genes









expression




cassette
primer
primer sequence (5′ > 3′)





nos::nptll-nos
forward
ATAATAACCGGTTGATCATGAGC




GGAGAATTAAGGG


 



reverse
ATAATAGCGGCCGCTAGTAACAT




AGATGACACCGCG


 


35S::aacC1-35S
forward
AATAGCGGCCGCGATTTAGTACT




GGATTTTGG


 



reverse
AATAACCGGTACCCACGAAGGAG




CATCGTGG


 


35S2::rbcS
forward
ATAATAACCGGTGCCCGGGGATC




TCCTTTGCC


 



reverse
ATAATAGCGGCCGCATGCATGTT




GTCAATCAATTGG


 


assu::tob
forward
TAATACCGGTAAATTTATTATGR




GTTTTTTTCCG


 



reverse
TAATGCGGCCGCTAAGGGCAGCC




CATACAAATGAAGC









The expression cassettes were further modified by introducing a GATEWAY® cloning cassette (Invitrogen) into the multicloning site of the respective pAUX vector following the manufacturer's protocol. In a recombination reaction, the cDNAs encoding the open reading frame sequences were transferred from the entry vector obtained as described in Example 4 to the GATEWAY®-enabled pAUX vector. Any combination of the regulatory elements with cDNA sequences of TrCHSa3, TrCHSc, TrCHSf, TrCHSh, TrBANa, TrLARa, TrLARb and TrLARc can be obtained. One typical example is given in FIG. 35 with expression cassettes for the nptII plant selectable marker, TrBANa, TrLARa and TrCHSa3.
Complete expression cassettes comprising any combination of regulatory elements and cDNA sequences to be introduced into the plant were then cut from the auxiliary vectors using the respective homing endonuclease and cloned into the respective restriction site on the binary vector backbone. After verification of the construct by nucleotide sequencing, the binary transformation vector comprising a number of expression cassettes was used to generate transgenic white clover plants.
EXAMPLE 6
Production by Agrobacterium-Mediated Transformation and Analysis of Transgenic White Clover Plants Carrying Chimeric White Clover TrCHSa3, TrCHSc, TrCHSf, TrCHSh, TrBANa, TrLARa, TrLARb and TrLARc Genes Involved in Flavonoid Biosynthesis
A set of binary transformation vectors carrying chimeric white clover genes involved in flavonoid biosynthesis, and more specifically condensed tannin biosynthesis to improve herbage quality and bloat-safety, were produced as detailed in Examples 4 and 5.
Agrobacterium-mediated gene transfer experiments were performed using these transformation vectors.
The production of transgenic white clover plants carrying the white clover TrCHSa3, TrCHSc, TrCHSf, TrCHSh, TrBANa, TrLARa, TrLARb and TrLARc cDNAs, either singly or in combination, is described here in detail.
Preparation of Agrobacterium 
Agrobacterium tumefaciens strain AGL-1 transformed with one of the binary vector constructs detailed in Example 6 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 I Erlenmeyer flask.
Preparation of White Clover Seeds
1 spoon of seeds (ca. 500) was placed into a 280 μm mesh size sieve and washed for 5 min under running tap water, taking care not to wash seeds out of sieve. In a laminar flow hood, seeds were transferred with the spoon into an autoclaved 100 ml plastic culture vessel. A magnetic stirrer (wiped with 70% EtOH) and ca. 30 ml 70% EtOH were added, and the seeds were stirred for 5 min. The EtOH was discarded and replaced by 50 ml 1.5% sodium hypochlorite. The seeds were stirred for an additional 45-60 min on a magnetic stirrer. The sodium hypochlorite was then discarded and the seeds rinsed 3 to 4 times with autoclaved ddH2O. Finally 30 ml of ddH2O were added, and seeds incubated over night at 10-15° C. in an incubator.
Agrobacterium-Mediated Transformation of White Clover
The seed coat and endosperm layer of the white clover seeds prepared as above were removed with a pair of 18 G or 21 G needles. The cotyledons were cut from the hypocotyl leaving a ca. 1.5 mm piece of the cotyledon stalk. The cotyledons were transferred to a petridish containing ddH2O. After finishing the isolation of clover cotyledons, ddH2O in the petridish was replaced with Agrobacterium suspension (diluted to an OD600=0.2-0.4). The petridish was sealed with its lid and incubated for 40 min at room temperature.
After the incubation period, each cotyledon was transferred to paper towel using the small dissection needle, dried and plated onto regeneration medium RM73. The plates were incubated at 25° C. with a 16 h light/8 h dark photoperiod. On day 4, the explants were transferred to fresh regeneration medium. Cotyledons transformed with Agrobacterium were transferred to RM73 containing cefotaxime (antibacterial agent) and gentamycin. The dishes were sealed with Parafilm and incubated at 25° C. under a 16/8 h photoperiod. Explants were subcultured every three weeks for a total of nine weeks onto fresh RM 73 containing cefotaxime and gentamycin. Shoots with a green base were then transferred to root-inducing medium RIM. Roots developed after 1-3 weeks, and plantlets were transferred to soil when the roots were well established.
This process is shown in detail in FIG. 36.
Preparation of Genomic DNA for Real-Time PCR and Analysis for the Presence of Transgenes
3-4 leaves of white clover plants regenerated on selective medium were harvested and freeze-dried. The tissue was homogenised 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 analysed on an agarose gel to check the yield and the quality of the genomic DNA.
Genomic DNA was analysed for the presence of the transgene by real-time PCR using SYBR Green chemistry. PCR primer pairs (Table 4) were designed using MacVector (Accelrys) or PrimerExpress (ABI). 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 endogenous genes in white clover were not detected.







TABLE 5







List of primers used for Real-time PCR analysis of white


clover plants transformed with chimeric white clover


genes involved in condensed tannin biosynthesis









construct
primer 1 (forward), 5′ −> 3′
primer 2 (reverse), 5′ −> 3′





pPZP221TrCHSa3
CATTTCATTTGGAGAGGACACGC
AACACGGTTTGGTGGATTTGC


 


pPZP221TrCHSc
TTGGAGAGGACACGCTGAAATC
ACAAGTTGGTGAGGGAATGCC


 


pPZP221TrCHSf
CATTTCATTTGGAGAGGACACGC
TCGTTGCCTTTCCCTGAGTAGG


 


pPZP221TrCHSh
TCATTTGGAGAGGACACGCTG
CGGTCACCATTTTTTTGTTGGAGG


 


pPZP221TrBANa
TTGGAGAGGACACGCTGAAATC
CAACAAAACCAGTGCCACC


 


pPZP221TrLARa
ATGACGCACAATCCCACTATCC
AGCCTTAGAAGAGAGAAGAGGTCC


 


pPZP221TrLARb
ATGACGCACAATCCCACTATCC
AGCCTTAGAAGAGAGAAGAGGTCC


 


pPZP221TrLARc
ATGACGCACAATCCCACTATCC
AGCCTTAGAAGAGAGAAGAGGTCC









5 μl of each genomic DNA sample was run in a 50 μl PCR reaction including SYBR Green on an ABI 7700 (Applied Biosystems) together with samples containing DNA isolated from wild type white clover plants (negative control), samples containing buffer instead of DNA (buffer control) and samples containing the plasmid used for transformation (positive plasmid control). Cycling conditions used were 2 min. at 50° C., 10 min. at 95° C. and then 40 cycles of 15 sec. at 95° C., 1 min. at 60° C.
Preparation of Genomic DNA and Analysis of DNA for Presence and Copy Number of Transgene by Southern Hybridisation Blotting
Genomic DNA for Southern hybridisation blotting was obtained from leaf material of white clover plants following the CTAB method. Southern hybridisation blotting experiments were performed following standard protocols as described in Sambrook et al. (1989). In brief, genomic DNA samples were digested with appropriate restriction enzymes and the resulting fragments separated on an agarose gel. After transfer to a membrane, a cDNA fragment representing a transgene or selectable marker gene was used to probe the size-fractionated DNA fragments. Hybridisation was performed with either radioactively labelled probes or using the non-radioactive DIG labelling and hybridisation protocol (Boehringer) following the manufacturer's instructions.
Plants were obtained after transformation with all chimeric constructs and selection on medium containing gentamycin. Details of plant analysis are given in Table 5 and FIGS. 37, 38 and 39.







TABLE 5







Transformation of white clover with binary transformation vectors comprising


cDNAs of white clover genes involved in condensed tannin biosyntheses,


selection and molecular analysis of regenerated plants.














cotyledons
selection



copy number


construct
transformed
into RIM
soil
QPCR-positive
Southern
range
















pPZP221-35S2::TrCHSa3
2358
135
32
23
n/d



pPZP221-35S2::TrCHSc
3460
89
41
27
n/d


pPZP221-35S2::TrCHSf
3931
113
44
27
n/d


pPZP221-35S2::TrCHSh
3743
79
37
30
n/d


pPZP221-35S2::TrBANa
2315
144
50
38
7
1 to 4


pPZP221-35S2::TrLARa
2487
88
45
38
n/d


pPZP221-35S2::TrLARb
3591
133
47
47
5
1 to 3


pPZP221-35S2::TrLARc
2835
96
32
29
n/d









REFERENCES


    Altschul, S. F., Gish, W., Miller, W., Myers, E. W., Lipman, D. J. (1990) “Basic local alignment search tool.” J. Mol. Biol. 215, 403-410.
    Frohman, M. A., Dush, M. K., Martin, G. R. (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, W., States, D. J. (1993) Identification of protein coding regions by database similarity search. Nature Genetics 3, 266-272.
    Goderis, I., De Bolle, M. F. C., Francois, I., Wouters, P. F. J., Broekaert, W. F., and Cammue, B. P. A. (2002) A set of modular plant transformation vectors allowing flexible insertion of up to six expression units. Plant Molecular Biology 50, 17-27.
    Hajdukiewicz P, Svab Z, Maliga P. (1994) The small, versatile pPZP family of Agrobacterium binary vectors for plant transformation. Plant Mol Biol. 25, 989-94.
    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


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.

## Claims

1. A purified or isolated nucleic acid comprising
(a) a sequence encoding a leucoanthocyanidine reductase (LAR) comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11, 13, and 15, or
(b) a variant of the sequence of paragraph (a), wherein the variant has at least 80% nucleic acid identity to any one of SEQ ID NOs: 11, 13 or 15, and encodes a polypeptide comprising the entirety of SEQ ID NOs: 12, 14, or 16, respectively.

2. The purified or isolated nucleic acid of claim 1, wherein the nucleic acid is a variant and the variant has at least 95% nucleic acid identity to any one of SEQ ID NOs: 11, 13 or 15.

3. The purified or isolated nucleic acid of claim 1, comprising a sequence selected from the group consisting of SEQ ID NOs: 11, 13 and 15 as the sequence encoding the LAR.

4. The purified or isolated nucleic acid of claim 1, further comprising a promoter sequence effective to direct expression of the nucleic acid sequence of (a), or (b) in a plant cell.

5. A plant cell, plant, plant seed or other plant part, comprising a nucleic acid construct, said construct comprising
a nucleic acid sequence selected from the group consisting of:
(a) a sequence encoding a leucoanthocyanidine reductase (LAR) comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11, 13, and 15, and
(b) a variant of the sequence of paragraph (a), wherein the variant has at least 80% nucleic acid identity to any one of SEQ ID NOs: 11, 13 or 15, and encodes a polypeptide comprising the entirety of SEQ ID NOs: 12, 14, or 16, respectively; and

a promoter sequence heterologous to the nucleic acid sequence effective to direct expression of the nucleic acid sequence in the plant cell, plant, plant seed or other plant part.

6. A method for modifying one or more processes selected from the group consisting of condensed tannin biosynthesis; protein binding; metal chelation; anti oxidation; UV-light absorption; and plant defense to a biotic stress in a plant, said method comprising
introducing into said plant an effective amount of a purified or isolated nucleic acid sequence comprising a sequence selected from the group consisting of:
(a) a sequence encoding a leucoanthocyanidine reductase (LAR) comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11, 13, and 15, and
(b) a variant of the sequence of paragraph (a), wherein the variant has at least 80% nucleic acid identity to any one of SEQ ID NOs: 11, 13 or 15, and encodes a polypeptide comprising the entirety of SEQ ID NO: 12, 14, or 16, respectively; and
expressing the nucleic acid sequence in the plant, whereby at least one of the processes is modified.

7. A method of modifying forage quality of a plant by disrupting protein foam and/or conferring protection from rumen pasture bloat, said method comprising
introducing into said plant an effective amount of a purified or isolated nucleic acid sequence comprising a sequence selected from the group consisting of:
(a) a sequence encoding a leucoanthocyanidine reductase (LAR) comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11, 13, and 15, and
(b) a variant of the sequence of paragraph (a), wherein the variant has at least 80% nucleic acid identity to any one of SEQ ID NOs: 11, 13 or 15, and encodes a polypeptide having LAR activity, and having at least 95% amino acid identity to SEQ ID NO: 12, 14, or 16, respectively; and
expressing the nucleic acid sequence in the plant, whereby the forage quality of the plant is modified.