AU2002213672A1 - Manipulation of plant life cycles and/or growth phases - Google Patents

Description

MANIPULATION OF PLANT LIFE CYCLES AND/OR GROWTH PHASES

The present invention relates to nucleic acids and nucleic acid fragments encoding amino acid sequences for proteins involved in the control of the transition to flowering in plants and the use thereof for the modification of plant life cycles and/or growth phases, flowering processes, flowering and plant architecture, and inflorescence and flower development.

Most plants have several growth phases. Following seed embryo germination, the plant apical meristem goes through a vegetative phase generating leaf primordia with axillary meristems. The axillary meristems will generate side branches or will rest dormant until apical dominance is removed. Upon receiving appropriate signals, the apical meristem switches to reproductive development (flowering). The switch is controlled by various physiological signals and genetic pathways that will coordinate flowering. The apical meristem switched from vegetative to reproductive phase will produce reproductive structures (inflorescences and flowers) instead of vegetative structures (leaves). This point is a critical developmental process in flowering plants.

The INDETERMINATE1 (id1) gene of maize controls the transition of flowering in this species by encoding a putative transcriptional regulator of flowering transition. An id1 mutation is the only mutation known to specifically and severely alter the ability of maize to undergo the transition to reproductive growth. Homozygous id1 maize mutants will produce many more leaves than wild-type maize plants. Maize id1 mutants remain in a prolonged vegetative growth state.

While nucleic acid sequences encoding some of the proteins involved in the control of plant life cycles and growth phases, flowering processes, flowering and plant architecture, and inflorescence and flower development have been isolated for certain species of plants, there remains a need for materials useful in the control of plant life cycles and growth phases, flowering processes, flowering and plant architecture, and inflorescence and flower development, in a wide range of plants, particularly in grasses and cereals including 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 and nucleic acid fragments encoding amino acid sequences for an ID1 protein from a ryegrass (Lolium) or fescue (Festuca) species, or a functionally active fragment or variant thereof.

The present invention also provides substantially purified or isolated nucleic acids and nucleic acid fragments encoding amino acid sequences for a class of proteins which are related to ID1. Such proteins are referred to herein as ID1-like.

The down-regulation or enhancement or ectopic expression or otherwise manipulation of id1 gene activity in grasses and cereals may alter plant life cycles and growth phases, for example it may alter the control of phase change, promote or reduce vegetative growth, delay or otherwise alter flowering, and/or alter floral organ and plant architecture e.g. vegetative-like inflorescences and flowers, enhanced branching, increased bushiness.

Manipulation of, for example, transition from vegetative phase to flowering phase or plant life cycles has significant consequences for a wide range of applications in plant production. For example, it has applications in delaying flowering in forage grasses and cereals thus reducing the formation of the less digestible stems and increasing herbage quality, in altering flowering time allowing early or late maturing grass and cereal crops, in delaying vegetative phase and thus increasing biomass production, in increasing branching and thus leading to enhanced bushiness, in altering plant size and leading to either higher or shorter plant stature, in blocking flowering and reducing the release of allergenic pollen, etc.

Methods of manipulating plant life cycles and growth phases, eg. the transition from the vegetative to the reproductive state, flowering and plant architecture in plants, including forage grasses and cereals, and grass species such as ryegrasses (Lolium species) and fescues (Festuca species), may facilitate the production of, for example, pasture grasses with enhanced or shortened or modified life cycles, enhanced or reduced or otherwise modified inflorescence and flower development, inhibited flowering (including non-flowering), modified flowering architecture (indeterminate and determinate), earlier or delayed flowering, enhanced or modified number of leaves, enhanced or reduced or otherwise modified number of reproductive shoots, enhanced persistence and improved herbage quality, enhanced seed and leaf yield, altered growth and development, leading to improved seed production, improved biomass production, improved pasture production, improved pasture quality, improved animal production and reduced environmental pollution (e.g. reduced pollen allergens, reduced nitrogenous waste).

The ryegrass (Lolium) or fescue (Festuca) species may be of any suitable type, including Italian or annual ryegrass, perennial ryegrass, tall fescue, meadow fescue and red fescue. Preferably the species is a ryegrass, more preferably perennial ryegrass (L. perenne). Perennial ryegrass (Lolium perenne L.) is a key pasture grass in temperate climates throughout the world.

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) may 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 an ID1 or ID1- like protein includes a nucleotide sequence selected from the group consisting of (a) sequences shown in Figures 1 , 2, 3, 4 and 5 hereto (Sequence ID Nos: 1 , 3, 5 and 7); (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) is capable of modifying the control of plant life cycles and/or growth phases, including flowering processes, and/or flowering or plant architecture 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 10 nucleotides, more preferably at least 15 nucleotides, most preferably at least 20 nucleotides. The nucleic acids or nucleic acid fragments encoding at least a portion of proteins involved in the control of plant life cycles and/or growth phases, including flowering processes, and/or flowering or plant architecture have been isolated and identified. The nucleic acids and 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 is well known in the art. Examples of sequence-dependent protocols include, but are not limited to, 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).

For example, genes encoding other proteins involved in the control of plant life cycles and/or growth phases, including flowering processes, and/or flowering or plant architecture, 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, the polymerase chain reaction may be performed on a library of cloned nucleic acid fragments wherein the sequence of one primer is derived from a nucleic acid or nucleic acid fragment 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. (1989J 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 ryegrass (Lolium) or fescue (Festuca) species, selected from the group consisting of ID1 and ID1 -like proteins; and functionally active fragments and variants thereof.

The ryegrass (Lolium) or fescue (Festuca) species may be of any suitable type, including Italian or annual ryegrass, perennial ryegrass, tall fescue, meadow fescue and red fescue. Preferably the species is a ryegrass, more preferably perennial ryegrass (L. perenne).

In a preferred embodiment of this aspect of the invention, the substantially purified or isolated ID1 or ID1-like polypeptide includes an amino acid sequence selected from the group consisting of sequences shown in Figures 1 , 2, 3, 4 and 6 hereto (Sequence ID Nos: 2, 4, 6 and 8) 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 ID1 or ID1-like. 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.

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 (SNP's), variations in single nucleotides between allelic forms of such nucleotide sequence, can be used as perfect markers or candidate genes for the given trait.

Applicants have identified a number of SNP's of the nucleic acids and nucleic acid fragments of the present invention. These are present in Figure 5 (Sequence ID Nos: 1 , 3, 5 and 7), which shows multiple alignments of nucleotide sequences of id1 nucleic acids of the present invention.

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 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 single nucleotide polymorphism (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 fragment 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 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 according to 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 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 forage and turf grass improvement, e.g. tagging QTLs for herbage quality traits, flowering intensity, flowering time, number of tillers, leafiness, bushiness, seasonal growth pattern, herbage yield, flower architecture, plant stature. 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 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 includes both cloning and expression vectors. Vectors are often recombinant molecules including 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, eg. derivatives of plant viruses; bacterial plasmids; derivatives of the Ti plasmid from Agrobacterlum tumefaciens, derivatives of the Ri plasmid from Agrobacterlum 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 (eg. monocotyledon or dicotyledon). Particularly suitable constitutive promoters include the Cauliflower Mosaic Virus 35S (CaMV 35S) promoter, 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) and the octopine synthase (ocs) 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 (hpli) gene, the phosphinothricin acetyltransferase (bar or pat) gene], and reporter genes (such as beta-glucuronidase (GUS) gene (gusA)]. 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, white clover, red clover, subterranean clover, alfalfa, eucalyptus, potato, sugarbeet) 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) and cereals such as maize (Zea mays) and rice (Oryza sativa), 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 lucerne (Medicago sativa).

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 Agrobacterlum 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 or a vector 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 or cereal species, more preferably a ryegrass (Lolium species) or fescue (Festuca species) or maize (Zea mays) or rice (Oryza sativa), even more preferably a ryegrass, most 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 lucerne (Medicago sativa).

The present invention also provides a plant, plant seed or other plant part derived from a plant cell of the present invention.

The present invention also provides a plant, plant seed or other plant part derived from a plant of the present invention.

In a further aspect of the present invention there is provided a method of modifying the control of plant life cycles and/or growth phases, including flowering processes, flowering, plant architecture, inflorescence or flower development, in a plant, said method including introducing into said plant an effective amount of a nucleic acid or nucleic acid fragment, a 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, plant life cycles and/or growth phases, including flowering processes, flowering, plant architecture, inflorescence or flower development may be increased, decreased or otherwise modified. For example, the number of leaves produced before flowering, the number of floral organs, the number of branches, the plant stature, the number of phytomers, the number of inflorescences and flowers, may be increased, decreased or otherwise modified. They may be increased or decreased, for example, by incorporating additional copies of a sense nucleic acid of the present invention or by incorporating an antisense nucleic acid of the present invention, respectively.

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

Figure 1 shows the nucleotide sequence of Lpldl (Sequence ID No: 1) and corresponding deduced amino acid sequence (Sequence ID No: 2). Figure 2 shows the nucleotide sequence of Lpld2 (Sequence ID No: 3) and corresponding deduced amino acid sequence (Sequence ID No: 4).

Figure 3 shows the nucleotide sequence of Lpld3 (Sequence ID No: 5) and corresponding deduced amino acid sequence (Sequence ID No: 6).

Figure 4 shows the nucleotide sequence of Lpld4 (Sequence ID No: 7) and corresponding deduced amino acid sequence (Sequence ID No: 8).

Figure 5 shows a nucleotide sequence alignment of the Lpldl , Lpld2, Lpld3 and Lpld4 nucleotide sequences (Sequence ID Nos: 1 , 3, 5 and 7, respectively) with conserved nucleotide positions (marked with black background) and SNPs and sequence differences (marked with white background).

Figure 6 shows the alignment of deduced amino acid sequences (Sequence ID Nos: 2, 4, 6 and 8, respectively) of the Lpldl , Lpld2, Lpld3 and Lpld4 nucleotide sequences with conserved amino acid residues (marked with black background) and two conserved Zinc Finger domains of transcriptional activators.

Figure 7 shows the alignment of deduced amino acid sequences (Sequence ID Nos: 2, 4, 6 and 8, respectively) from the Lpldl , Lpld2, Lpld3 and Lpld4 nucleotide sequences with conserved amino acid residues (marked with black background) and the maize Id1 (Sequence ID No: 9) and the potato ID1-like protein PCP1 (Sequence ID No: 10).

Figure 8 shows plasmid maps of ID1 homologue cDNAs Lpldl , Lpld2, Lpld3 and Lpld4 isolated from perennial ryegrass (Lolium perenne).

Figure 9 shows plasmid maps of plant transformation vectors with perennial ryegrass ID1 homologue cDNA Lpld4 sequences in sense and antisense orientation under control of CaMV 35S promoter.

Figure 10 shows Southern hybridisation analysis of perennial ryegrass genomic DNA using ryegrass ID1 homologue cDNA Lpld4 as hybridisation probe (Lane 1. uncut genomic DNA; lane 2. EcoRI digested genomic DNA; lane 3. Hindlll digested genomic DNA; lane 4. Kpnl digested genomic DNA).

Figure 11 shows northern hybridisation analysis revealing expression patterns of perennial ryegrass ID1 homologue cDNA Lpld4 in different perennial ryegrass plant organs and developmental stages (Lane 1. 3 day old shoots; lane 2. 3 day old roots; lane 3. 10 day old shoots; lane 4. 10 day old roots; lane 5. mature leaves; lane 6. leaves from flowering stem).

Figure 12 shows the regeneration of transgenic tobacco plants carrying chimeric sense and antisense perennial ryegrass ID1 homologue genes.

EXAMPLE 1

A perennial ryegrass (Lolium perenne) cDNA library was prepared from mRNA isolated from 8 -10 day old seedlings. Total RNA was isolated using the Trizol method (Gibco-BRL, USA) following the manufacturers' instructions. A cDNA library was generated using the UniZAP-cDNA^R Synthesis Kit according to the manufacturer's protocol (Stratagene Cloning Systems, La Jolla, CA, USA). 50,000 plaques were screened with a ryegrass ID1 PCR fragment generated using oligonucleotides designed to the maize id1 gene. Positive plaques were selected and converted into plasmids according to the protocol provided by Stratagene. Upon conversion, cDNA inserts were contained in the plasmid vector pBluescript (Figure 8). Plasmid DNA was prepared (Qiagen, Germany) according to the protocol provided by Qiagen and cDNA inserts sequenced using dye- terminator sequencing reactions and analyzed using an Applied Biosystems ABI 3700 sequence analyser.

EXAMPLE 2

DNA and protein sequence analyses

The cDNA clones encoding ID1 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 ANGIS 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 results from the deduced amino acid sequence alignments are shown on Figure 7.

EXAMPLE 3

Development of transformation vectors containing chimeric genes with ID1 cDNA sequences from perennial ryegrass

To alter the expression of ID1 gene activity in transgenic plants through antisense and/or sense suppression technology and for over-expression or ectopic a set of sense and antisense transformation vectors was produced.

cDNA fragments were generated from the cDNA clone of Lpld4 using the restriction enzymes BamHI and Sphl. The sense construct was generated by direct cloning of this fragment into the transformation vector pDH51 , which was digested with the same enzymes. The antisense construct was obtained by removing the 3' overhang following Sphl digestion to produce a blunt end. The fragment was then digested with BamHI and this fragment was cloned into pDH51 digested with BamHI and Smal. Transformation vectors containing this 750bp region of the Lpld4 cDNA in sense and antisense orientations under the control of the CaMV 35S promoter were generated (Figure 9).

EXAMPLE 4

Production of transgenic tobacco plants carrying chimeric ID1 homologue genes from perennial ryegrass

A set of transgenic tobacco plants carrying chimeric sense and antisense ID1 homologue genes from perennial ryegrass were produced.

pDH51 -based transformation vectors with Lpld4 cDNA comprising a 750bp fragment in sense and antisense orientations under the control of the CaMV 35S promoter were generated (Figure 9).

Direct gene transfer experiments to tobacco protoplasts were performed using these transformation vectors (Table 1).

TABLE 1

Production of transgenic tobacco calli carrying chimeric perennial ryegrass ID1 homologue genes (in sense and antisense orientation) from direct gene transfer to protoplasts

The production of transgenic tobacco plants carrying the perennial ryegrass Lpld4 cDNA under the control of the constitutive CaMV 35S promoter is described here in detail. Isolation of mesophyll protoplasts from tobacco shoot cultures

2 to 4 fully expanded leaves of a 6 week-old shoot culture were placed under sterile conditions (work in laminar flow hood, use sterilized forceps, scalpel and blades) in a 9 cm plastic culture dish containing 12 ml enzyme solution [1.0% (w/v) cellulase "Onozuka" R10 and 1.0% (w/v) Macerozyme^® R10]. The leaves were wetted thoroughly with enzyme solution and the mid-ribs removed. The leaf halves were cut into small pieces and incubated overnight (14 to 18 h) at 25°C in the dark without shaking

The protoplasts were released by gently pipetting up and down, and the suspension poured through a 100 μm stainless steel mesh sieve on a 100 ml glass beaker. The protoplast suspension was mixed gently, distributed into two 14 ml sterile plastic centrifuge tubes and carefully overlayed with 1 ml W5 solution.

After centrifugation for 5 min. at 70g (Clements Orbital 500 bench centrifuge, swing-out rotor, 400 rpm), the protoplasts were collected from the interphase and transferred to one new 14 ml centrifuge tube. 10 ml W5 solution were added, the protoplasts resuspended by gentle tilting the capped tube and pelleted as before.

The protoplasts were resuspended in 5 to 10 ml W5 solution and the yield determined by counting a 1 :10 dilution in a haemocytometer.

Direct gene transfer to protoplasts using polyethylene glycol The protoplasts were pelleted [70g (Clements Orbital 500 bench centrifuge,

400 rpm) for 5 min.] and resuspended in transformation buffer to a density of 1.6 x 10⁶ protoplasts/ml. Care should be taken to carry over as little as possible W5 solution into the transformation mix. 300 μ\ samples of the protoplast suspension (ca. 5 x 10⁵ protoplasts) were aliquotted in 14 ml sterile plastic centrifuge tubes, 30 μ\ of transforming DNA were added. After carefully mixing, 300 μ\ of PEG solution were added and mixed again by careful shaking. The transformation mix was incubated for 15 min. at room temperature with occasional shaking. 10 ml W5 solution were gradually added, the protoplasts pelleted [70g (Clements Orbital 500 bench centrifuge, 400 rpm) for 5 min.] and the supernatant removed. The protoplasts were resuspended in 0.5 ml K3 medium and ready for cultivation. Culture of protoplasts, selection of transformed lines and regeneration of transgenic tobacco plants

Approximately 5 x 10⁵ protoplasts were placed in a 6 cm petri dish. 4.5 ml of a pre-warmed (melted and kept in a water bath at 40 to 45°C) 1 :1 mix of K3:H medium containing 0.6% SeaPlaque™ agarose were added and, after gentle mixing, allowed to set.

After 20 to 30 min the dishes were sealed with Parafilm^® and the protoplasts were cultured for 24 h in darkness at 24°C, followed by 6 to 8 days in continuous dim light (5 μmol m^"2 s^"\ Osram L36 W/21 Lumilux white tubes), where first and multiple cell divisions occur. The agarose containing the dividing protoplasts was cut into quadrants and placed in 20 ml of A medium in a 250 ml plastic culture vessel. The corresponding selection agent was added to the final concentration of 50 mg/l kanamycin sulphate (for npt2 expression) or 25 mg/l hygromycin B (for hph expression) or 20 mg/l phosphinotricin (for bar expression). Samples were incubated on a rotary shaker with 80 rpm and 1.25 cm throw at 24°C in continuous dim light.

Resistant colonies were first seen 3 to 4 weeks after protoplast plating, and after a total time of 6 to 8 weeks protoplast-derived resistant colonies (when 2 to 3 mm in diameter) were transferred onto MS morpho medium solidified with 0.6% (w/v) agarose in 12-well plates and kept for the following 1 to 2 weeks at 24°C in continuous dim light (5 μmol m^"2 s^"1, Osram L36 W/21 Lumilux white tubes), where calli proliferated, reached a size of 8 to 10 mm, differentiated shoots that were rooted on MS hormone free medium leading to the recovery of transgenic tobacco plants (Table 1 and Figure 12).

EXAMPLE 5

Genomic organization of perennial ryegrass ID1 homologue genes

Genomic DNA from perennial ryegrass was digested with the following restriction enzymes EcoRI, Hindlll and Kpnl. Southern blot analysis was then performed according to standard protocols (Ausubel et al. (1994) Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience). The probe used for screening this blot was a 750bp gene specific fragment of the Lpld4 cDNA obtained by restriction digestion with BamHI and Sphl. Lpld4 exists as a single copy gene in the genome of perennial ryegrass (Figure 10).

EXAMPLE 6

Expression of perennial ryegrass ID1 homologue genes

A northern hybridisation analysis with RNA samples isolated from perennial ryegrass at different developmental stages was performed to determine patterns of organ and developmental expression of ryegrass ID1 genes. Total RNA was extracted from the following tissues using Trizol reagent (GibcoBRL, USA) three day old shoots and roots, ten day old shoots and roots, mature leaves and leaves taken from the flowering stem. Northern blot analysis was performed according to according to standard protocols (Ausubel et al. (1994) Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience). The probe used for screening this blot was a 750 bp gene specific fragment of the Lpld4 cDNA obtained by restriction digestion with BamHI and Sphl. Lpld4 is most strongly expressed in three-day old roots and mature leaves (Figure 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.

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 an acknowledgment that they form part of the common general knowledge in the relevant art.

Claims (14)

1. A substantially purified or isolated nucleic acid or nucleic acid fragment encoding an amino acid sequence for an ID1 protein from a ryegrass (Lolium) or fescue (Festuca) species, or a functionally active fragment or variant thereof.

2. A nucleic acid or nucleic acid fragment according to Claim 1 , wherein said ryegrass is perennial ryegrass (Lolium perenne).

3. A nucleic acid or nucleic acid fragment according to Claim 1 , including a nucleotide sequence selected from the group consisting of (a) sequences shown in Figures 1 , 2, 3, 4 and 5 hereto (Sequence ID Nos: 1 , 3, 5 and 7) ; (b) complements of the sequences 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).

4. A construct including a nucleic acid or nucleic acid fragment according to Claim 1.

5. A vector including a nucleic acid or nucleic acid fragment according to Claim 1.

6. A vector according to Claim 5, further including a promoter and a terminator, said promoter, nucleic acid or nucleic acid fragment and terminator being operatively linked.

7. A plant cell, plant, plant seed or other plant part, including a construct according to Claim 4 or a vector according to Claim 5.

8. A plant, plant seed or other plant part derived from a plant cell or plant according to Claim 7.

9. A method of modifying plant life cycles and/or growth phases in a plant, said method including introducing into said plant an effective amount of a nucleic acid or nucleic acid fragment according to Claim 1 , a construct according to Claim 4, and/or a vector according to Claim 5.

10. A method according to claim 9 wherein said plant life cycle and/or growth phase is selected from the group consisting of flowering processes, flowering and plant architecture, and inflorescence and flower development.

11. Use of a nucleic acid or nucleic acid fragment according to Claim 1 , and/or nucleotide sequence information thereof, and/or single nucleotide polymorphisms thereof as a molecular genetic marker.

12. A substantially purified or isolated polypeptide from a ryegrass (Lolium) or fescue (Festuca) species, selected from the group consisting of ID1 and ID1-like proteins; and functionally active fragments and variants thereof.

13. A polypeptide according to Claim 12, wherein said ryegrass is perennial ryegrass (Lolium perenne).

14. A polypeptide according to Claim 12, wherein said polypeptide includes an amino acid sequence selected from the group of sequences shown in

Figures 1 , 2, 3, 4 and 6 hereto (Sequence ID Nos: 2, 4, 6 and 8); and functionally active fragments and variants thereof.

SEQUENCE LISTING

<110> Molecular Plant Breeding Nominees Ltd

<120> Manipulation of plant life cycles and/or growth phases

<130> M80199534

<160> 10

<170> Patentln version 3.1

<210> 1

<211> 2588

<212> DNA

<213> Lolium perenne

<220>

<221> misc_feature

<222> (1903) .. (1903)

<223> Any nucleotide

<400> 1 ggcacgagaa ggagatgaaa gaaatcagac aacaaccttc ttcttagcct tcctcatccc 60 cacttctatc tcttccttca gttgcaggga gcaaggagga agagctagca gcagcagttg 120 gcagagagag tgagcgaagg gcaaagaaga agaagaaagg aacactgcca atccaaggaa 180 accaaagagg agaagctttg ttcttgtgct tggagttggt gtaagagggt attaaggcga 240 tggcatcgaa ctcatcggca gcagcggcgg cggccttctt cgggatgaga gatggggacc 300 agcaagacca gatgaagccg ctgataacac agcaccagca gctggcagcg gtggcactgc 360 ccggcgccgc gcctgcggcg tccagccaac acggcgcacc gccggcggcg gcgccaccgg 420 ccaagaagaa gaggactcta ccagacccag acgcggaggt gatcgcgctt tctcccaaga 480 cgctgatggc gactaaccgg ttcgtgtgcg aggtgtgcca gaagggtttc cagcgcgagc 540 agaacctgca gctacaccgt cgcgggcaca acctcccctg gaagctgaag cagaagaacc 600 cgaaccaggt gcagcggcgt cgcgtgtacc tgtgcccgga ggtgacgtgc gtccaccacg 660 acccgtcgcg cgccctgggc gatctcaccg ggatcaagaa gcacttctgc cgcaagcacg 720 gcgagaagaa gtggaagtgc gacaagtgct ccaagcgcta cgccgtgcag tccgactgga 780 aggcgcactc caagatctgc ggcacacgcg agtaccgctg cgactgcggc accctcttct 840 ccaggaggga cagcttcatc acccacaggg ccttctgcga cgcactggcg caggagagcg 900 ccaggctgcc gccgacgagc ctcagcaccc tcaccagcca cctctacggt gccaccaacg 960 ccggcaacat ggcgctcagc ctgtcccagg tgggatccca cctcaactcc accatgcagc 1020 acgacggcgg ccaccaccac cacccgtccc cagacctcct ccgcctcggt ggcggcggcg 1080 gcagcagcag catcgccgcg cggctcgacc acctcctgtc tccgacaggc gcgtccgcgt 1140 tccgcgcgaa tcagcagcag ccccagccgg ccttcttcct caacgcggct ccggggcagg 1200 acttcggtga cgatcagggg ggcaacggac cccactcttc atgcagtcaa agcccttcca 1260 cggctcatgc agttccggac cttcagggca acggcgcggc ggccggccat gggctcttca 1320 acctgggctt ctttgccaac aacggcaata gctccgggtc aagtcacgag catgcaagcc 1380 agggcatgat gagcaacgat cagttcagcg gcggagctgg tggaggcggc aacggctctg 1440 aggtgtcggc cgcggggatc tttggcggca actttgttgg tggggatcac atggcgcagg 1500 ctgggatgta caacgaccag gcggctatgc tgccgcagat gtctgccacc gcgctgctcc 1560 agaaggccgc gcagatgggc gcgacctcca gccccaacgg cgcggcgtcc atgttcaggg 1620 gcttcgccgg ctcctcgccg cagctgcgac aggcggcacc tcagcacatg gaccagaacg 1680 aggcgaacct caacgagctg atgaactctc tggctgctgg tggcggcgtc aatgccgccg 1740 gaatgttcgg cggcgccaac ggtggcσccg gcgcggggat gtttgatcct aggatgtgcg 1800 acatggaσca gcacgaggtg aagttcagcc agggcggagg cggtgttggt gccaaccctc 1860 ctcctggtgg tggtggtggt ggtgccgccg cgccgccaac atnacgcgga cttcctcggc 1920 gtggcgaagc ggcatcgtgc ccgggatatc gactccaaga ggtgaccaca accaaagcag 1980 cagcgacatg agctccctgg aggccgagat gaagtcggct tcgtcctaca acgggggccg 2040 gatggcatga tcgagagctt acacaagσcg taagctccca tcatcacagt gagtctaaga 2100 cttgaagcaa cctggaatta gcatgcatgc atatatgaga ctgagatgag taaggttaat 2160 tagcgagtta gcaactaagt tgatcgcatg agtatggtcg catgcatgca tgggataagg 2220 ctagctagct agctctccat ggggccatga agactggagt accgtgaaag ggagaggcct 2280 cctctgagac taattaagta attagctagt gσgagtgaac gcatgcatgt gatggcccta 2340 gcttggtcgt cgaggagttg gtgtcagtgt cggtcctgtt gtttattttc cttccttgta 2400 agtggatctt tgctgggtta agtatagttc tggagataga accaataagg ctattttagt 2460 tttgtagcta gccgaaatgg gatgttgggg agctgaactt gcccctgaaa ctccttcttc 2520 tgttcctttt attacccttg tagcgaaaga atgctgctct tacagtgcaa aaaaaaaaaa 2580 aaaaaact 2588

<210> 2

<211> 583

<212> PRT

<213> Lolium perenne

<220>

<221> MISC_FEATURE <222> (555).. (555) <223> Any amino acid

<400> 2

Met Ala Ser Asn Ser Ser Ala Ala Ala Ala Ala Ala Phe Phe Gly Met 1 5 10 15

Arg Asp Gly Asp Gin Gin Asp Gin Met Lys Pro Leu lie Thr Gin His 20 25 30

Gin Gin Leu Ala Ala Val Ala Leu Pro Gly Ala Ala Pro Ala Ala Ser 35 40 45 Ser Gin His Gly Ala Pro Pro Ala Ala Ala Pro Pro Ala Lys Lys Lys 50 55 60

Arg Thr Leu Pro Asp Pro Asp Ala Glu Val lie Ala Leu Ser Pro Lys 65 70 75 80

Thr Leu Met Ala Thr Asn Arg Phe Val Cys Glu Val Cys Gin Lys Gly 85 90 95

Phe Gin Arg Glu Gin Asn Leu Gin Leu His Arg Arg Gly His Asn Leu 100 105 110

Pro Trp Lys Leu Lys Gin Lys Asn Pro Asn Gin Val Gin Arg Arg Arg 115 120 125

Val Tyr Leu Cys Pro Glu Val Thr Cys Val His His Asp Pro Ser Arg 130 135 140

Ala Leu Gly Asp Leu Thr Gly He Lys Lys His Phe Cys Arg Lys His 145 150 155 160

Gly Glu Lys Lys Trp Lys Cys Asp Lys Cys Ser Lys Arg Tyr Ala Val 165 170 175

Gin Ser Asp Trp Lys Ala His Ser Lys He Cys Gly Thr Arg Glu Tyr 180 185 190

Arg Cys Asp Cys Gly Thr Leu Phe Ser Arg Arg Asp Ser Phe He Thr 195 200 205

His Arg Ala Phe Cys Asp Ala Leu Ala Gin Glu Ser Ala Arg Leu Pro 210 215 220

Pro Thr Ser Leu Ser Thr Leu Thr Ser His Leu Tyr Gly Ala Thr Asn 225 230 235 240

Ala Gly Asn Met Ala Leu Ser Leu Ser Gin Val Gly Ser His Leu Asn 245 250 255

Ser Thr Met Gin His Asp Gly Gly His His His His Pro Ser Pro Asp 260 265 270

Leu Leu Arg Leu Gly Gly Gly Gly Gly Ser Ser Ser He Ala Ala Arg 275 280 285

Leu Asp His Leu Leu Ser Pro Thr Gly Ala Ser Ala Phe Arg Ala Asn 290 295 300

Gin Gin Gin Pro Gin Pro Ala Phe Phe Leu Asn Ala Ala Pro Gly Gin 305 310 315 320 Asp Phe Gly Asp Asp Gin Gly Gly Asn Gly Pro His Ser Ser Cys Ser 325 330 335

Gin Ser Pro Ser Thr Ala His Ala Val Pro Asp Leu Gin Gly Asn Gly 340 345 350

Ala Ala Ala Gly His Gly Leu Phe Asn Leu Gly Phe Phe Ala Asn Asn 355 360 365

Gly Asn Ser Ser Gly Ser Ser His Glu His Ala Ser Gin Gly Met Met 370 375 380

Ser Asn Asp Gin Phe Ser Gly Gly Ala Gly Gly Gly Gly Asn Gly Ser 385 390 395 400

Glu Val Ser Ala Ala Gly He Phe Gly Gly Asn Phe Val Gly Gly Asp 405 410 415

His Met Ala Gin Ala Gly Met Tyr Asn Asp Gin Ala Ala Met Leu Pro 420 425 430

Gin Met Ser Ala Thr Ala Leu Leu Gin Lys Ala Ala Gin Met Gly Ala 435 440 445

Thr Ser Ser Pro Asn Gly Ala Ala Ser Met Phe Arg Gly Phe Ala Gly 450 455 460

Ser Ser Pro Gin Leu Arg Gin Ala Ala Pro Gin His Met Asp Gin Asn 465 470 475 480

Glu Ala Asn Leu Asn Glu Leu Met Asn Ser Leu Ala Ala Gly Gly Gly 485 490 495

Val Asn Ala Ala Gly Met Phe Gly Gly Ala Asn Gly Gly Pro Gly Ala 500 505 510

Gly Met Phe Asp Pro Arg Met Cys Asp Met Asp Gin His Glu Val Lys 515 520 525

Phe Ser Gin Gly Gly Gly Gly Val Gly Ala Asn Pro Pro Pro Gly Gly 530 535 540

Gly Gly Gly Gly Ala Ala Ala Pro Pro Thr Xaa Arg Gly Leu Pro Arg 545 550 555 560

Arg Gly Glu Ala Ala Ser Cys Pro Gly Tyr Arg Leu Gin Glu Val Thr 565 570 575

Thr Thr Lys Ala Ala Ala Thr 580 <210> 3

<211> 2220

<212> DNA

<213> Lolium perenne

<400> 3 ggcacgagct cgtgccgaat tcggcacgag aaaagtatga σaaggagatg aaagaaatca 60 gacaacaacc ttcttcttag ccttcctcat ccccacttct atctcttcct tcagttgcag 120 ggagcaagga ggaagagcta gcagcagcag ttggcagaga gagtgagcga agggcaaaga 180 agaagaagaa aggaacactg ccaatccaag gaaaccaaag aggagaagct ttgttcttgt 240 gcttggagtt ggtgtaagag ggtattaagg cgatggcatc gaactcatcg gcagcagcgg 300 cggcggcctt cttcgggatg agagatgggg accagcaaga ccagatgaag ccgctgataa 360 cacagcacca gcagctggca gcggtggcac tgcccggcgc cgcgcctgcg gcgtccagcc 420 aacacggcgc accgccggcg gcggcgccac cggccaagaa gaagaggact ctaccagacc 480 cagacgcgga ggtgatcgcg ctttctccca agacgctgat ggcgactaac cggttcgtgt 540 gcgaggtgtg ccagaagggt ttccagcgcg agcagaacct gcagctacac cgtcgcgggc 600 acaacctccc ctggaagctg aagcagaaga acccgaacca ggtgcagcgg cgtcgcgtgt 660 acctgtgccc ggaggtgacg tgcgtccacc acgacccgtc gcgcgccctg ggcgatctca 720 ccgggatcaa gaagcacttc tgccgcaagc acggcgagaa gaagtggaag tgcgacaagt 780 gctccaagcg ctacgccgtg cagtccgact ggaaggcgca ctccaagatc tgcggcacac 840 gcgagtaccg ctgcgactgc ggcaccctct tctccaggag ggacagcttc atcacccaca 900 gggccttctg cgacgcactg gcgcaggaga gcgccaggct gccgccgacg agcctcagca 960 ccctcaccag ccacctctac ggtgccacca acgccggcaa catggcgctc agcctgtccc 1020 aggtgggatc ccacctcaac tccaccatgc agcacgacgg cggccaccac caccacccgt 1080 ccccagacct cctccgcctc ggtggcggcg gcggcagcag cagcatcgcc gcgcggctcg 1140 accacctcct gtctccgaca ggcgcgtccg cgttccgcgc gaatcagcag cagccccagc 1200 cggccttctt cctcaacgσg gctccggggc aggacttcgg tgacgatcag gggggcaacg 1260 gaccccattc cttcatgcag tcaaaagccc tttccacggc ctcatgcagc tcccggacct 1320 tcagggcaaσ ggcgcgggcg ggccgggccc atgggctctt caacctgggc ttctttgcca 1380 acaacggcaa tagctccggg tcaagtcacg agcatgcaag ccagggcatg atgagcaacg 1440 atcagttcag cggcggagσt ggtggaggcg gcaacggctc tgaggtgtcg gccgcgggga 1500 tctttggcgg caactttgtt ggtggggatc acatggcgca ggctgggatg tacaacgacc 1560 aggcggctat gctgccgcag atgtctgcca ccgcgctgct ccagaaggcc gcgcagatgg 1620 gcgcgacctc cagσcccaac ggcgcggcgt ccatgttcag gggcttcgcc ggctcctcgc 1680 cgcagctgcg acaggcggca cctcagcaca tggaccagaa cgaggcgaac ctcaacgagc 1740 tgatgaactc tctggctgct ggtggcggcg tcaatgccgc cggaatgttc ggcggcgcca 1800 acggtggccc cggcgcgggg atgtttgatc ctaggatgtg cgacatggac cagcacgagg 1860 tgaagttcag ccagggcgga ggcggtgttg gtgccaaccc tgctgctggt ggtggtggtg 1920 gtggtggcgg cggcggcggc gacatgacgc gggacttcct cggcgtgggc ggaggcggca 1980 tcgtgcgcgg gatatcgact ccaagaggtg accacaacca aagcagcagc ggacatgagc 2040 tccctggagg ccgaaatgaa gtcggcttcg tcctacaacg ggggccggat ggcatgatcg 2100 agagcttacc caagccgtaa gctcccatca tcccagtgag tttaagactt gaagcaacct 2160 ggaattagca tgcatgcata tatgagactg agatgagtaa ggttaattag cgagttagca 2220

<210> 4

<211> 615

<212> PRT

<213> Lolium perenne

<400> 4

Met Ala Ser Asn Ser Ser Ala Ala Ala Ala Ala Ala Phe Phe Gly Met 1 5 10 15

Arg Asp Gly Asp Gin Gin Asp Gin Met Lys Pro Leu He Thr Gin His 20 25 30

Gin Gin Leu Ala Ala Val Ala Leu Pro Gly Ala Ala Pro Ala Ala Ser 35 40 45

Ser Gin His Gly Ala Pro Pro Ala Ala Ala Pro Pro Ala Lys Lys Lys 50 55 60

Arg Thr Leu Pro Asp Pro Asp Ala Glu Val He Ala Leu Ser Pro Lys 65 70 75 80

Thr Leu Met Ala Thr Asn Arg Phe Val Cys Glu Val Cys Gin Lys Gly 85 90 95

Phe Gin Arg Glu Gin Asn Leu Gin Leu His Arg Arg Gly His Asn Leu 100 105 110

Pro Trp Lys Leu Lys Gin Lys Asn Pro Asn Gin Val Gin Arg Arg Arg 115 120 125

Val Tyr Leu Cys Pro Glu Val Thr Cys Val His His Asp Pro Ser Arg 130 135 140

Ala Leu Gly Asp Leu Thr Gly He Lys Lys His Phe Cys Arg Lys His 145 150 155 160

Gly Glu Lys Lys Trp Lys Cys Asp Lys Cys Ser Lys Arg Tyr Ala Val 165 170 175

Gin Ser Asp Trp Lys Ala His Ser Lys He Cys Gly Thr Arg Glu Tyr 180 185 190 Arg Cys Asp Cys Gly Thr Leu Phe Ser Arg Arg Asp Ser Phe He Thr 195 200 205

His Arg Ala Phe Cys Asp Ala Leu Ala Gin Glu Ser Ala Arg Leu Pro 210 215 220

Pro Thr Ser Leu Ser Thr Leu Thr Ser His Leu Tyr Gly Ala Thr Asn 225 230 235 240

Ala Gly Asn Met Ala Leu Ser Leu Ser Gin Val Gly Ser His Leu Asn 245 250 255

Ser Thr Met Gin His Asp Gly Gly His His His His Pro Ser Pro Asp 260 265 270

Leu Leu Arg Leu Gly Gly Gly Gly Gly Ser Ser Ser He Ala Ala Arg 275 280 285

Leu Asp His Leu Leu Ser Pro Thr Gly Ala Ser Ala Phe Arg Ala Asn 290 295 300

Gin Gin Gin Pro Gin Pro Ala Phe Phe Leu Asn Ala Ala Pro Gly Gin 305 310 315 32_0

Asp Phe Gly Asp Asp Gin Gly Gly Asn Gly Pro His Ser Phe Met Gin 325 330 335

Ser Lys Ala Leu Ser Thr Ala Ser Cys Ser Ser Arg Thr Phe Arg Ala 340 345 350

Thr Ala Arg Ala Gly Arg Ala His Gly Leu Phe Asn Leu Gly Phe Phe 355 360 365

Ala Asn Asn Gly Asn Ser Ser Gly Ser Ser His Glu His Ala Ser Gin 370 375 380

Gly Met Met Ser Asn Asp Gin Phe Ser Gly Gly Ala Gly Gly Gly Gly 385 390 395 400

Asn Gly Ser Glu Val Ser Ala Ala Gly He Phe Gly Gly Asn Phe Val 405 410 415

Gly Gly Asp His Met Ala Gin Ala Gly Met Tyr Asn Asp Gin Ala Ala 420 425 430

Met Leu Pro Gin Met Ser Ala Thr Ala Leu Leu Gin Lys Ala Ala Gin 435 440 445

Met Gly Ala Thr Ser Ser Pro Asn Gly Ala Ala Ser Met Phe Arg Gly 450 455 460 Phe Ala Gly Ser Ser Pro Gin Leu Arg Gin Ala Ala Pro Gin His Met 465 470 475 480

Asp Gin Asn Glu Ala Asn Leu Asn Glu Leu Met Asn Ser Leu Ala Ala 485 490 495

Gly Gly Gly Val Asn Ala Ala Gly Met Phe Gly Gly Ala Asn Gly Gly 500 505 510

Pro Gly Ala Gly Met Phe Asp Pro Arg Met Cys Asp Met Asp Gin His 515 520 525

Glu Val Lys Phe Ser Gin Gly Gly Gly Gly Val Gly Ala Asn Pro Ala 530 535 540

Ala Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Asp Met Thr Arg 545 550 555 560

Asp Phe Leu Gly Val Gly Gly Gly Gly He Val Arg Gly He Ser Thr 565 570 575

Pro Arg Gly Asp His Asn Gin Ser Ser Ser Gly His Glu Leu Pro Gly 580 585 590

Gly Arg Asn Glu Val Gly Phe Val Leu Gin Arg Gly Pro Asp Gly Met 595 600 605

He Glu Ser Leu Pro Lys Pro 610 615

<210> 5

<211> 2474

<212> DNA

<213> Lolium perenne

<220>

<221> misc_feature

<222> (571).. (571)

<223> Any nuσleotide

<400> 5 gtcaagggcg agcgagaaga. ccaccacagc tagcgaagca gcgcaagatc agatagataa 60 gagagacacg caggtgaaag gaaatttgct agccgaaggc tagcctacaa aaagggaaag 120 ctttgcctgc ttccggcgag agtagctagc ttcaacggcg atggcgtcca actcatcggc 180 ggcggctgtg gcggcgttgt ttggaattag ggatggccac cagcaggacc agatcaagcc 240 gctgcttcσg ctgcagcagc agcaccacca gcσcgccctc gcgccgccca gcgcggtggc 300 ggcgggaccg gaccagccgg ctgcggcggt gccaccggtg aagaagaaga gaaccatgcc 360 tgaccctgac gctgaggtga tcgcgctgtc gcccaagacg ctgatggcga cgaaccggtt 420 cgtctgcgag gtgtgcaaca aggggttcca gcgggagcag aacctgcagc tgcaccggcg 480 cggccacaac ctgccgtgga agctgaagca gaaggacccc aaccaggtgc agcggcggcg 540 ggtgtacctc tgcccggagc cgacgtgcgt ncaccacgag cccggccgcg cgctgggcga 600 ccacaccggc atcaagaagc acttσtgccg caagcacggc gagaagaaat ggaagtgcga 660 caagtgcgcc aagcgctacg ccgtgcagtc ggactggaag gcgcactcca aggtctgcgg 720 cacccgcgag taccgctgcg actgcggcac cctattctcc cggagggaca gcttcatcac 780 ccacagggcc ttctgcgacg ccctcaccca ggagagcgcg cgcctggcac ccgccgccca 840 cctctacggc ggcgccacgg ccgctgccaa catggcgctc agcctctctc aggtgggatc 900 ctccttccag gacgtccacg gccagtacca ccagcaggca tcctcggacc tcctσcgctt 960 cggcggcggt ggcggcggcg gcggcatggc tgcccgcctc gaccacctcc tctcgtcgtc 1020 caccttccgc aacctgccgc ctccccaagc accggcgccg ttccacctcg gccagacgca 1080 gcaggagttc ggcgacggga acaacaacgg cccgcacgcg ttcctgcagg gcaagccgtt 1140 ccacggcctc atgcagctcc cggacctcca gggcaacggc tccgggggca cggcgtcgtc 1200 ggctcccggt ctcttcaacc tcggcggcta catcgccaac aagcgccaac agctccggcg 1260 gtacctcagc cacggccacg ccagccaggg gcacatggcc aacaatgacc agatcagtga 1320 aggagccggt ggtgccggtt ctgagaactc cggcgcggcg ttcttcaacg ccgcggccgg 1380 tgggaacttc tccggtggcg accaccacca ggttgttgct cccgctggga tgtacaacga 1440 gcagcaggcc atggcgatgc tgσcgcagat gtcggccacg gccctgctcc agaaggcggc 1500 acagatgggc tccagcacca gcgcggacgg cggcggctcc tcctccatat tcagcggttt 1560 cctgggccag tccgaccagc agggtcgcgc gcccagcatg gtggaccagg gaatgcatct 1620 ccagagcσtg atgaactcgc tggccggcgg gagcaacgga ggcgggatσt ttggaggcgg 1680 cggcaacggc cggggtatga ttgatccgag actgtatgac atggaccagc acgaggtgaa 1740 atttagccag cagggccgtg ggggcagcgg cggcggcggt ggagacgtga caagggactt 1800 tctcggcgtt ggtggcagag gggacgtgat cagagggatg tcagtggcaa gaggggagca 1860 ccacagcggc ggcggcggcg acatgagctt cttggaggcc gagatgaagt cggcctcgtc 1920 gcccttcaat ggaggcagga tgcagtgatg atgagcttaa ttttctttag aaaggcatga 1980 agctggctta agttcgttcc gatcgagtcg tgaacctgag aaccatatgc atggaagaag 2040 ctgtttactt acaactttgt ttacatgtca gcacaaatgt tttgacttct gggagσaagg 2100 ttggaagggt atgcatcaat gcatgagctt cttcttccac caattcaaga aaggtaaaac 2160 gactcaaatc attagactag atcttatatt agcggcagt cttcctgtca atgttttgtt 2220 accgcagttg aatattatta aatgtaattg gattaattca tgccggctag ggagagatcg 2280 ttgcatatac cgtaaggttt ttgtagctgg caatgtgagg gatggttaga acttgcgcac 2340 cccaaactca atcttttttt cttttcttac aaataatgac attgctgtag cccgaatggg 2400 tggtatgaat tttctttttc cgtaaaaaaa aaaaaaaaaa aaaaaaaact cgagactagt 2460 tctcgtgccg aatt 2474

<210> 6

<211> 595

<212> PRT

<213> Lolium perenne

<400> 6

Met Ala Ser Asn Ser Ser Ala Ala Ala Val Ala Ala Leu Phe Gly He 1 5 10 15

Arg Asp Gly His Gin Gin Asp Gin He Lys Pro Leu Leu Pro Leu Gin 20 25 30

Gin Gin His His Gin Pro Ala Leu Ala Pro Pro Ser Ala Val Ala Ala 35 40 45

Gly Pro Asp Gin Pro Ala Ala Ala Val Pro Pro Val Lys Lys Lys Arg 50 55 60

Thr Met Pro Asp Pro Asp Ala Glu Val He Ala Leu Ser Pro Lys Thr 65 70 75 80

Leu Met Ala Thr Asn Arg Phe Val Cys Glu Val Cys Asn Lys Gly Phe 85 90 95

Gin Arg Glu Gin Asn Leu Gin Leu His Arg Arg Gly His Asn Leu Pro 100 105 110

Trp Lys Leu Lys Gin Lys Asp Pro Asn Gin Val Gin Arg Arg Arg Val 115 120 125

Tyr Leu Cys Pro Glu Pro Thr Cys Val His His Glu Pro Gly Arg Ala 130 135 140

Leu Gly Asp His Thr Gly He Lys Lys His Phe Cys Arg Lys His Gly 145 150 155 160

Glu Lys Lys Trp Lys Cys Asp Lys Cys Ala Lys Arg Tyr Ala Val Gin 165 170 175

Ser Asp Trp Lys Ala His Ser Lys Val Cys Gly Thr Arg Glu Tyr Arg 180 185 190

Cys Asp Cys Gly Thr Leu Phe Ser Arg Arg Asp Ser Phe He Thr His 195 200 205

Arg Ala Phe Cys Asp Ala Leu Thr Gin Glu Ser Ala Arg Leu Ala Pro 210 215 220

Ala Ala His Leu Tyr Gly Gly Ala Thr Ala Ala Ala Asn Met Ala Leu 225 230 235 240 Ser Leu Ser Gin Val Gly Ser Ser Phe Gin Asp Val His Gly Gin Tyr 245 250 255

His Gin Gin Ala Ser Ser Asp Leu Leu Arg Phe Gly Gly Gly Gly Gly 260 265 270

Gly Gly Gly Met Ala Ala Arg Leu Asp His Leu Leu Ser Ser Ser Thr 275 280 285

Phe Arg Asn Leu Pro Pro Pro Gin Ala Pro Ala Pro Phe His Leu Gly 290 295 300

Gin Thr Gin Gin Glu Phe Gly Asp Gly Asn Asn Asn Gly Pro His Ala 305 310 315 320

Phe Leu Gin Gly Lys Pro Phe His Gly Leu Met Gin Leu Pro Asp Leu 325 330 335

Gin Gly Asn Gly Ser Gly Gly Thr Ala Ser Ser Ala Pro Gly Leu Phe 340 345 350

Asn Leu Gly Gly Tyr He Ala Asn Lys Arg Gin Gin Leu Arg Arg Tyr 355 360 365

Leu Ser His Gly His Ala Ser Gin Gly His Met Ala Asn Asn Asp Gin 370 375 380

He Ser Glu Gly Ala Gly Gly Ala Gly Ser Glu Asn Ser Gly Ala Ala 385 390 395 400

Phe Phe Asn Ala Ala Ala Gly Gly Asn Phe Ser Gly Gly Asp His His 405 410 415

Gin Val Val Ala Pro Ala Gly Met Tyr Asn Glu Gin Gin Ala Met Ala 420 425 430

Met Leu Pro Gin Met Ser Ala Thr Ala Leu Leu Gin Lys Ala Ala Gin 435 440 445

Met Gly Ser Ser Thr Ser Ala Asp Gly Gly Gly Ser Ser Ser He Phe 450 455 460

Ser Gly Phe Leu Gly Gin Ser Asp Gin Gin Gly Arg Ala Pro Ser Met 465 470 475 480

Val Asp Gin Gly Met His Leu Gin Ser Leu Met Asn Ser Leu Ala Gly

4l o85c 4 /i o9n0 4 /l9a5c.

Gly Ser Asn Gly Gly Gly He Phe Gly Gly Gly Gly Asn Gly Arg Gly 500 505 510 Met He Asp Pro Arg Leu Tyr Asp Met Asp Gin His Glu Val Lys Phe 515 520 525

Ser Gin Gin Gly Arg Gly Gly Ser Gly Gly Gly Gly Gly Asp Val Thr 530 535 540

Arg Asp Phe Leu Gly Val Gly Gly Arg Gly Asp Val He Arg Gly Met 545 550 555 560

Ser Val Ala Arg Gly Glu His His Ser Gly Gly Gly Gly Asp Met Ser 565 570 575

Phe Leu Glu Ala Glu Met Lys Ser Ala Ser Ser Pro Phe Asn Gly Gly 580 585 590

Arg Met Gin 595

<210> 7

<211> 1887

<212> DNA

<213> Lol^~i"um—perenne

<220>

<221> misc_feature

<222> (1712) .. (1712)

<223> Any nucleotide

<400> 7 tcggcacgag tgcacatgca ccatcgcccc gcacggccgc gagatctgat cgattttgca 60 gcggctagct gaggattccg tggaaatttc agcagcgagc tggtggattt ttgtagctag 120 ctggggtcga tcgagatgat gctcaaggat ctggcggcaa ttcagcagca gcagcagcag 180 cagctggccc tggccgcggc ggcggacgag aaσatgtσσa acctcacctc cgcgtccggc 240 gaccagaσca gcgtctcctc ccaccctctc ccgcctcctt ccaagaggaa gcgcagcctc 300 ccgggaaacc cagaσcccga cgcggaggtg atcgcgctgt cgccgcggtc gctcatggcc 360 acgaaccgct acgtgtgcga gatctgcggc aagggcttcc agcgggaσca gaaσctgcag 420 ctgcaccgcc gcggccacaa cctgccctgg aagctcaagc agcgcaaccc caacgaggtg 480 gtgcgcaaga aggtctacgt ctgcccggag cccggctggg tgcaσcacga ccgcgcccgc 540 gcgctcggcg acctcacggg gatcaagaag caσttcagcc gaaagcacgg cgagaagaag 600 tggaagtgcg acaagtgcgc caagcgatac gccgtgcagt ccgactggaa ggcgcactcc 660 aaggtctgcg gcaccaggga gtaccgctgc gactgcggca ccctcttctc aaggcgggac 720 agcttcatca cgcacagggc tttctgcgac gcgctggcgg aggagagcgc cagggccgtc 780 gcggtggatc cggggatgct ctactctcac agcggcggcg gcagcgcggg gtttcagatg 840 ccggccgtca tggacgcgtc gcacccgctg ggcgccgggc acgggctcat ccaagaactg 900 tgcctcaaga gggagcagca gcagcaacat caacaacagt tcgcgcagcc gtggctatcc 960 gagcagcagc agatggagat ggccagcgag ggcgcccccc cggggatgtt tggcacggcg 1020 aggatggacc aggagttcaa cgggagcagc acgccggaga gcagcacgca gccggcgggc 1080 atgggcttcg cgtccttctc gtcgcccgcg gcggcggggc cgtccgcctc cgggtccacg 1140 cacatgtcag ccaccgcgct gctccagaag gcggcgcaga tgggcgcgac gctgagccgt 1200 ccgtcgggcc agggccagat ggcgccgagt accctcagca gcagcagcgt tggcggcgcc 1260 gccaacaata atgcaccggc tgccgctact accactaaca gcgcgacgac aagcactgcc 1320 atcggcgctg gattcgcgσa cacgttcgag gcgccagcgc acttcggggt gcaccagaga 1380 tccaattcca gcagtcgcaa tgccggcaat ggcgccaccg gggccggagg tggtgcaatg 1440 ccgggggcgg caacgacggc caaacgaggg acttcctggg gctgcgggca ttctcccacg 1500 gcgacatact cagcatggca gggttcgatc cctgcatgcc cacctcggcc tctgcgtccg 1560 cggcagcgta cgatcagcaa gggcaccaga gcaccgagcc atggcacggc tagcaaggta 1620 cagtactagt gatgaacata gctatcagct ggctagctcg ccatcgatga gctgaaccgt 1680 gtcaattcga tgcaccatgc catggcctaa tnttcatcgc ctctggatgc ccagcattgc 1740 tctcgatcat atatgtcaag tttctcatgg ggtaagtggt cgatatccag ttcgtttttg 1800 ttgcacgcca ctcagtttga tcttcttttc ggaggaaata atgaactgtt caaattcatg 1860 taaaaaaaaa aaaaaaaaaa aaactcg 1887

<210> 8

<211> 497

<212> PRT

<213> Lolium perenne

<400> 8

Met Met Leu Lys Asp Leu Ala Ala He Gin Gin Gin Gin Gin Gin Gin 1 5 10 15

Leu Ala Leu Ala Ala Ala Ala Asp Glu Asn Met Ser Asn Leu Thr Ser 20 25 30

Ala Ser Gly Asp Gin Thr Ser Val Ser Ser His Pro Leu Pro Pro Pro 35 40 45

Ser Lys Arg Lys Arg Ser Leu Pro Gly Asn Pro Asp Pro Asp Ala Glu 50 55 60

Val He Ala Leu Ser Pro Arg Ser Leu Met Ala Thr Asn Arg Tyr Val 65 70 75 80

Cys Glu He Cys Gly Lys Gly Phe Gin Arg Asp Gin Asn Leu Gin Leu 85 90 95

His Arg Arg Gly His Asn Leu Pro Trp Lys Leu Lys Gin Arg Asn Pro 100 105 HO Asn Glu Val Val Arg Lys Lys Val Tyr Val Cys Pro Glu Pro Gly Trp 115 120 125

Val His His Asp Arg Ala Arg Ala Leu Gly Asp Leu Thr Gly He Lys 130 135 140

Lys His Phe Ser Arg Lys His Gly Glu Lys Lys Trp Lys Cys Asp Lys 145 150 155 160

Cys Ala Lys Arg Tyr Ala Val Gin Ser Asp Trp Lys Ala His Ser Lys 165 170 175

Val Cys Gly Thr Arg Glu Tyr Arg Cys Asp Cys Gly Thr Leu Phe Ser 180 185 190

Arg Arg Asp Ser Phe He Thr His Arg Ala Phe Cys Asp Ala Leu Ala 195 200 205

Glu Glu Ser Ala Arg Ala Val Ala Val Asp Pro Gly Met Leu Tyr Ser 210 215 220

His Ser Gly Gly Gly Ser Ala Gly Phe Gin Met Pro Ala Val Met Asp 225 230 235 240

Ala Ser His Pro Leu Gly Ala Gly His Gly Leu He Gin Glu Leu Cys 245 250 255

Leu Lys Arg Glu Gin Gin Gin Gin His Gin Gin Gin Phe Ala Gin Pro 260 265 270

Trp Leu Ser Glu Gin Gin Gin Met Glu Met Ala Ser Glu Gly Ala Pro 275 280 285

Pro Gly Met Phe Gly Thr Ala Arg Met Asp Gin Glu Phe Asn Gly Ser 290 295 300

Ser Thr Pro Glu Ser Ser Thr Gin Pro Ala Gly Met Gly Phe Ala Ser 305 310 315 320

Phe Ser Ser Pro Ala Ala Ala Gly Pro Ser Ala Ser Gly Ser Thr His 325 330 335

Met Ser Ala Thr Ala Leu Leu Gin Lys Ala Ala Gin Met Gly Ala Thr 340 345 350

Leu Ser Arg Pro Ser Gly Gin Gly Gin Met Ala Pro Ser Thr Leu Ser 355 360 365

Ser Ser Ser Val Gly Gly Ala Ala Asn Asn Asn Ala Pro Ala Ala Ala 370 375 380 Thr Thr Thr Asn Ser Ala Thr Thr Ser Thr Ala He Gly Ala Gly Phe 385 390 395 400

Ala His Thr Phe Glu Ala Pro Ala His Phe Gly Val His Gin Arg Ser 405 410 415

Asn Ser Ser Ser Arg Asn Ala Gly Asn Gly Ala Thr Gly Ala Gly Gly 420 425 430

Gly Ala Met Pro Gly Ala Ala Thr Thr Ala Lys Arg Gly Thr Ser Trp 435 440 445

Gly Cys Gly His Ser Pro Thr Ala Thr Tyr Ser Ala Trp Gin Gly Ser 450 455 460

He Pro Ala Cys Pro Pro Arg Pro Leu Arg Pro Arg Gin Arg Thr He 465 470 475 480

Ser Lys Gly Thr Arg Ala Pro Ser His Gly Thr Ala Ser Lys Val Gin 485 490 495

Tyr

<210> 9

<211> 509

<212> PRT

<213> potato

<400> 9

Met Ser He Val Thr Cys Glu Lys Ala Ala Ala Ser Leu Ser Ser Ser 1 5 10 15

Ser Asn Met Asn Asn Asp Thr Asn Gly Ala Phe Cys Tyr Thr Pro Gin 20 25 30

His Gin Leu Val Thr Pro Gin Tyr Gin Asn Pro Pro Gin Gin He Lys 35 40 45

Lys Lys Arg Asn Gin Pro Gly Asn Pro Asp Pro Glu Ala Glu Val He 50 55 60

Ala Leu Ser Pro Lys Thr Leu Val Ala Ala Asn Arg Phe Phe Cys Glu 65 70 75 80

He Cys Asn Lys Gly Phe Gin Arg Asp Gin Asn Leu Gin Leu His Arg 85 90 95

Arg Gly His Asn Leu Pro Trp Lys Leu Lys Lys Arg Glu Asn Lys Glu 100 . 105 110 Val Val Arg Lys Lys Val Tyr He Cys Pro Glu Ser Ser Cys Val Hiε 115 120 125

His Asp Pro Ser Arg Ala Leu Gly Asp Leu Thr Gly He Lys Lys His 130 135 140

Phe Ser Arg Lys His Gly Glu Lys Lys Trp Lys Cys Glu Lys Cys Ser 145 150 155 160

Lys Arg Tyr Ala Val Gin Ser Asp Cys Lys Ala His Phe Lys Thr Cys 165 170 175

Gly Thr Arg Glu Tyr Lys Cys Glu Cys Gly Thr He Phe Ser Arg Arg 180 185 190

Asp Ser Phe He Thr His Arg Ala Phe Cys Glu Thr Leu Ala Met Glu 195 200 205

Ser Ala Arg Ser Val He Asn Gly Arg Asn Pro Thr He Phe Ser Pro 210 215 220

Gin-Leu Asn Leu Gin Phe Gin Gin Pro His Phe Phe Asn_ Ser His Glu 225 230 235 240

Gin He Gin Ala Thr Thr Phe Pro Met Lys Lys Glu Gin Gin Ser Ser 245 250 255

Asp Phe Arg His He Glu He Pro Pro Trp Leu He Thr Thr Asn Ser 260 265 270

Gin Pro Phe Gin Leu Gly Ala He Asn His Gly Pro Ser Pro Arg Ser 275 280 285

Asn Phe Ser Ser Ser Ser He Phe Pro Ala Thr Thr Arg Leu Asp Gin 290 295 300

Gin Tyr Thr Gin Ser Gly His Lys Asp Leu Asn Leu His His Pro Asn 305 310 315 320

Pro Asn Leu Arg Gly Pro Thr Leu Gly Tyr Asp Ser Thr Gly Glu Ser 325 330 335

Gly Ala Val Ser Pro Val His He Ser Ala Thr Arg Leu Leu Gin Lys 340 345 350

Ala Ala Gin Phe Gly Ala Thr He Ser Asn Lys Ala Ser Ala Val Thr 355 360 365

Ala Thr Ala Ala Tyr Thr Gly Thr Val Lys He Pro His Asn Thr His 370 375 380 Val Ser Val Thr Ser Thr Asp Ser Ala Thr Lys Gin Thr His Gin Lys 385 390 395 400

Leu Ser Ser Arg Glu Asp Leu Thr Ser He Thr Gly Pro Ala Asn He 405 410 415

Ser Gly He Met Thr Ser Phe Ser Asn Gly Phe Asp Gly Ser Thr Met 420 425 430

Phe Glu Asp Ala He Leu Phe Gly Gly Phe Asn Asn Leu Asn Ser Lys 435 440 445

Lys Glu Asp Glu Glu Glu Asp Gin Gin Leu Tyr Phe Asn Gly Ser Met 450 455 460

Asn Glu Glu Asp His He Leu Thr Lys Asp Phe Leu Gly Leu Lys Pro 465 470 475 480

Leu Ser His Thr Asp Asp He Phe Asn He Ala Ala Leu Val Asn Thr 485 490 495

Glu Pro His His Phe Lys Asn His Lys Thr Trp Gin Ser 500 505

<210> 10

<211> 436

<212> PRT

<213> maize

<400> 10

Met Gin Met Met Met Leu Ser Asp Leu Ser Ser Asp Asp His Glu Ala 1 5 10 15

Thr Gly Ser Ser Ser Tyr Gly Gly Asp Met Ala Ser Tyr Ala Leu Ser 20 25 30

Pro Leu Phe Leu Ala Pro Ala Ala Ser Ala Thr Ala Pro Leu Pro Pro 35 40 45

Pro Pro Gin Pro Pro Ala Glu Glu Leu' Thr Asn Lys Gin Ala Ala Gly 50 55 60

Gly Gly Lys Arg Lys Arg Ser Gin Pro Gly Asn Pro Asp Pro Gly Ala 65 70 75 80

Glu Val He Ala Leu Ser Pro Arg Thr Leu Val Ala Thr Asn Arg Phe 85 90 - 95

Val Cys Glu He Cys Asn Lys Gly Phe Gin Arg Asp Gin Asn Leu Gli 100 105 . 110 Leu His Arg Arg Gly His Asn -Leu Pro Trp Lys Leu Arg Gin Arg Ser 115 120 125

Ser Leu Val Val Pro Ser Ser Ser Ala Ala Ala Gly Ser Gly Gly Arg 130 135 140

Gin Gin Gin Gin Gin Gly Glu Ala Ala Pro Thr Pro Pro Arg Lys Arg 145 150 155 160

Val Tyr Val Cys Pro Glu Pro Thr Cys Val His His Asp Pro Ala Arg 165 170 175

Ala Leu Gly Asp Leu Thr Gly He Lys Lys His Phe Ser Arg Lys His 180 185 190

Gly Glu Lys Arg Trp Cys Cys Glu Arg Cys Gly Lys Arg Tyr Ala Val 195 200 205

Gin Ser Asp Trp Lys Ala His Val Lys Gly Cys Gly Thr Arg Glu Tyr 210 215 220

Arg Cys Asp Cys Gly He Leu Phe Ser Arg Lys Asp Ser Leu Leu Thr 225 230 235 240

His Arg Ala Phe Cys Asp Ala Leu Ala Glu Glu Ser Ala Arg Leu Leu 245 250 255

Ala Ala Ala Ala Asn Asn Gly Ser Thr He Thr Thr Thr Ser Ser Ser 260 265 270

Asn Asn Asn Asp Leu Leu Asn Ala Ser Asn Asn He Thr Pro Leu Phe 275 280 285

Leu Pro Phe Ala Ser Ser Pro Pro Pro Val Val Val Ala Ala Ala Gin 290 295 300

Asn Pro Asn Asn Thr Leu Phe Phe Leu His Gin Glu Leu Ser Pro Phe 305 310 315 320

Leu Gin Pro Arg Val Thr Met Gin Gin Gin Pro Ser Pro Tyr Leu Asp 325 330 335

Leu His Met His Val Asp Ala Ser He Val Thr Thr Thr Gly Gly Leu 340 345 350

Ala Asp Gly Thr Pro Val Ser Phe Gly Leu Ala Leu Asp Gly Ser Val 355 360 365

Ala Thr Val Gly His Arg Arg Leu Thr Arg Asp Phe Leu Gly Val Asp 370 375 380 Gly Gly Gly Arg Gin Val Glu Glu Leu Gin Leu Pro Leu Cys Ala Thr 385 390 395 400

Ala Ala Ala Ala Gly Ala Ser Arg Thr Ala Ser Cys Ala Thr Asp Leu 405 410 415

Thr Arg Gin Cys Leu Gly Gly Arg Leu Pro Pro Val Asn Glu Thr Trp 420 425 430

Ser His Asn Phe 435