Plant phosphomevalonate kinases

The invention relates to nucleic acids which encode plant polypeptides with the biological activity of phosphomevalonate kinases, to the polypeptides encoded by them and their use as targets for herbicides and their use for identifying novel, herbicidally active compounds, and to methods for finding modulators of these polypeptides.

Unwanted plant growth can be prevented by using herbicides. The demands made on herbicides with regard to their efficacy, costs and environmental compatibility have been steadily increasing. There exists therefore a demand for new substances which can be developed into potent new herbicides. In general, it is usual to search for such new lead structures in greenhouse tests. However, such tests are laborious and expensive. Accordingly, the number of substances which can be tested in the greenhouse is limited.

Advantageous targets for herbicides are searched for in essential biosynthetic pathways. Thus, the biosynthesis of isoprenoids in plants leads, inter alia, to the synthesis of carotenoids and of the side chains of plastoquinone and of chlorophyll. These products are essential for the photosynthetic growth of plants. The inhibition of one step in this biosynthetic pathway leads to the termination of a plant's growth. Moreover, plant hormones such as gibberellic acid, abscisic acid and brassinosteroids and membrane components (phytosterols), which are also essential for the plant's growth, are formed from isoprenoids.

Isopentyl diphosphate (IPP) is the branching point from which the widest range of isoprenoids are formed. The production of IPP is therefore a critical point in plant metabolism. In plants, IPP is produced via two different metabolic pathways in different compartments. In the endoplasmic reticulum (ER) and in the cytosol, IPP synthesis proceeds via the classic acetate/mevalonate metabolic pathway as it also proceeds in the animal organism. In contrast, IPP is synthesized in chloroplasts via the alternative glyceraldehyde phosphate/pyruvate metabolic pathway. Both metabolic pathways are essential since various isoprenoid metabolites are formed in the different compartments. Moreover, the degree to which the two metabolic pathways are autonomous or to which an exchange of metabolites takes place between the compartments has not been elucidated as yet (Heintze et al., 1990, Kleinig, 1989). (See References section below for full citation to these and other references referred to herein).

Clomazone is a known herbicidal compound which reduces the carotenoid and chlorophyll content in the leaf. For a long time it has been assumed that clomazone acts via the inhibition of the isoprenoid metabolic pathway. Norman et al. (1990) had demonstrated that the site of action would have to be between mevalonate and geranylgeranyl pyrophosphate. This would establish one of the interposed five enzymes, one of which is phosphomevalonate kinase, as the site of action. Somewhat more recent works by Weimer et al. (1992) and Rodney Croteau (1992) suggest, however, that the site of action of clomazone would be found elsewhere.

Within the context of the present invention, an Arabidopsis thaliana cv. Columbia cDNA has been isolated with homology to phosphomevalonate kinase, hereinbelow abbreviated to PMVK, from Saccharomyces cerevisiae (FIG. 1 ). It was possible to induce this gene in Arabidopsis thaliana cv. Columbia by treatment with the herbicide chlorsulfuron (10 g/ha).

The homology between the Saccharomyces cerevisiae PMVK (=ERG8) and the cDNA isolated from A. thaliana amounts to 44% similarity or 35% identity (see FIG. 1, Bestfit with Wisconsin Package Version 10.1). (ERG8 is the name of the gene encoding phosphomevalonate kinase in yeast ( S cerevisiae )). This corresponds for example to the homology between the Saccharomyces cerevisiae mevalonate kinase and the Arabidopsis thaliana mevalonate kinase with a similarity of 45% and an identity of 35%. The function was detected for the Arabidopsis thaliana mevalonate kinase by complementation of the corresponding mutant from Saccharomyces cerevisiae. Moreover, the cDNA isolated within the context of the present invention shows 69% identity with a partial PMVK sequence from Pinus radiate in accordance with SEQ ID NO:5, which is of interest for modifying the isoprenoid content, isoprenoid composition and isoprenoid metabolism of plants (WO 00/36 081). Further partial cDNAs from plants ( Medicago trunculata, Accession Number AA660847, see SEQ ID NO:3 and Gossypium hirsutum, Accession Number Al727861, see SEQ ID NO:4) have been isolated as putative PMVKs. Various Arabidopsis spp. sequences (ESTs and genomic sequences) which correspond to the PMVK sequence isolated herein or to parts thereof can be found in databases from various sequencing projects, however, no information is given on the function or importance of these sequences or sequence fragments.

For the first time, the present invention now provides the complete cDNA sequence of a plant phosphomevalonate kinase and describes its use, or the use of the polypeptides encoded thereby, for identifying new herbicidal active compounds.

Subject-matter of the present invention are therefore nucleic acids which encode complete plant phosphomevalonate kinases, with the exception of the partial nucleic acid sequences from Medicago trunculata in accordance with SEQ ID NO:3, Gossypium hirsutum in accordance with SEQ ID NO:4 and Pinus radiata in accordance with SEQ ID NO:5.

Subject-matter of the present invention are, in particular, nucleic acids which encode the Arabidopsis thaliana phosphomevalonate kinase.

Subject-matter of the present invention are very particularly nucleic acids which encode the Arabidopsis thaliana phosphomevalonate kinase and are described under SEQ ID NO:1 and/or encode a polypeptide in accordance with SEQ ID NO:2 or fragments thereof.

FIG. 1 is a determination of the homology between the A. thaliana phosphomevalonate kinase according to the invention in accordance with SEQ ID NO:2 and the known S. cerevisiae phosphomevalonate kinase (BESTFIT) by means of Bestfit (Wisconsin Package Version 10.1 (GCG)). The similarity is 44% and the identity 35%.

SEQ ID NO:1 Nucleic acid sequence encoding A. thaliana phosphomevalonate kinase.

SEQ ID NO:2 Amino acid sequence of the A. thaliana phosphomevalonate kinase.

SEQ ID NO:3 Nucleic acid fragment from Medicago trunculata (putative PMVK) of Accession Number AA 660847.

SEQ ID NO:4 Nucleic acid fragment from Gossypium hirsutum (putative PMVK) of Accession Number Al 727861.

SEQ ID NO:5 Nucleic acid fragment from Pinus radiata (encoding PMVK in accordance with WO 00/36081).

The nucleic acids according to the invention are, in particular, single-stranded or double-stranded deoxyribonucleic acids (DNA) or ribonucleic acids (RNA).

Preferred embodiments are fragments of the genomic DNA, which may contain introns, and cDNAs.

The nucleic acids according to the invention are preferably DNA fragments which correspond to the cDNA of Arabidopsis plants.

The nucleic acids according to the invention especially preferably encompass a sequence selected from amongst

a) the sequence in accordance with SEQ ID NO:1,

b) sequences which encode a polypeptide which encompasses the amino acid sequence in accordance with SEQ ID NO: 2,

c) part sequences of the sequences defined under a) or b) which have a length of at least 14 base pairs,

d) sequences which hybridize with the sequences defined under a) or b) at a hybridization temperature of 35-52° C.,

e) sequences which have at least 70% identity, preferably 85% identity, especially preferably 90% identity, very especially preferably 95% identity, with the sequences defined under a),

f) sequences which have at least 70% identity, preferably 80% identity, especially preferably 90% identity, very especially preferably 95% identity, with the sequences defined under b),

g) sequences which are complementary to the sequences defined under a) or b), and

h) sequences which, owing to the degeneracy of the genetic code, encode the same amino acid sequence as the sequences defined under a) to f).

A cDNA molecule with the sequence in accordance with SEQ ID NO:1 constitutes a very especially preferred embodiment of the nucleic acids according to the invention.

The term “complete” phosphomevalonate kinase as used in the present context describes the phosphomevalonate kinase which is encoded by a complete coding region of a transcription unit starting with the ATG start codon and encompassing all information-bearing exon regions of the gene present in the organism of origin and encoding phosphomevalonate kinase, and the signals required for correct termination of transcription.

The term “gene” as used in the present context refers to a section from the genome of a cell which section is responsible for the synthesis of a polypeptide chain.

The term “to hybridize” as used in the present context describes the process in which a single-stranded nucleic acid molecule undergoes base pairing with a complementary strand. In this manner, for example, DNA fragments which encode phosphomevalonate kinases which exhibit the same or similar properties as the kinase with the amino acid sequence in accordance with SEQ ID NO: 2 can be isolated from plants other than Arabidopsis, starting from the sequence information disclosed herein.

The term “cDNA” as used in the present context refers to the single- or double-stranded copy of an RNA molecule and, being a copy of biologically active mRNA, is free from introns, i.e. all coding regions of a gene are present in connected form. Hybridization conditions are calculated by approximation using the following formula:

Melting temperature Tm=81.5° C.+16.6 log [ c (Na + )]+0.41(% G+C ))−500/ n

(Lottspeich and Zorbas, 1998).

In this formula, c is the concentration and n the length of the hybridizing sequence segment in base pairs. For a sequence >100 bp, the term 500/n does not apply. At the highest stringency, washing is effected at a temperature of 5-15° C. below Tm and an ionic strength of 15 mM Na + (corresponds to 0.1×SSC). If an RNA probe is used for hybridization, the melting point is 10-15° C. higher.

Preferred hybridization conditions are indicated hereinbelow:

Hybridization solution: DIG Easy Hyb (Roche)

Hybridization temperature: 35-52° C., preferably 42° C. (DNA-DNA), or 50° C. (DNA-RNA).

1. Wash step: 2×SSC, twice 5 minutes at room temperature;

2. Wash step: twice 15 minutes in 1×SSC, at 50° C.; preferably 0.5×SSC, at 65° C.; especially preferably 0.2×SSC, at 65° C.

The degree of identity of the nucleic acids is preferably determined with the aid of the program NCBI BLASTN Version 2.0.14. (Altschul et al., 1997).

Subject-matter of the present invention are furthermore DNA constructs which encompass a nucleic acid according to the invention and a homologous or heterologous promoter.

The term “homologous promoter” as used in the present context refers to a promoter which controls the expression of the gene in question in the organism of origin.

The term “heterologous promoter” as used in the present context refers to a promoter which has properties other than the promoter which controls the expression of the gene in question in the organism of origin.

The choice of heterologous promoters depends on whether pro- or eukaryotic cells or cell-free systems are used for expression. Examples of heterologous promoters are the cauliflower mosaic virus 35S promoter for plant cells, the alcohol dehydrogenase promoter for yeast cells, and the T3-, T7- or SP6 promoters for prokaryotic cells or cell-free systems.

Subject-matter of the present invention are furthermore vectors comprising a nucleic acid according to the invention, a regulatory region according to the invention or a DNA construct according to the invention. Vectors which can be used are all the phages, plasmids, phagemids, phasmids, cosmids, YACs, BACs, artificial chromosomes or particles which are suitable for particle bombardment that are used in molecular biology laboratories.

Preferred vectors are pBIN (Bevan, 1984) and its derivatives for plant cells, pFL61 (Minet et al., 1992) or, for example, the p4XXprom. series of vectors (Mumberg et al.) for yeast cells, pSPORT vectors (Life Technologies) for bacterial cells, lambdaZAP (Stratagene) for phages or Gateway vectors (Life Technologies) for various expression systems in bacterial cells or in baculovirus.

Subject-matter of the present invention are also host cells comprising a nucleic acid according to the invention, a DNA construct according to the invention or a vector according to the invention.

The term “host cell” as used in the present context refers to cells which do not naturally comprise the nucleic acids according to the invention.

Suitable host cells are not only prokaryotic cells, preferably E. coli, but also eukaryotic cells such as cells of Saccharomyces cerevisiae, Pichia pastoris, insects, plants, frog oocytes and mammalian cell lines.

Subject-matter of the present invention are furthermore polypeptides with the biological activity of phosphomevalonate kinases which are encoded by the nucleic acids according to the invention.

The term “polypeptides” as used in the present context relates not only to short amino acid chains which are usually termed peptides, oligopeptides or oligomers, but also to longer amino acid chains which are usually termed proteins. It encompasses amino acid chains which can be modified either by natural processes such as posttranslational processing or by chemical prior-art methods. Such modifications can occur at various positions and repeatedly in a polypeptide, such as, for example, at the peptide backbone, at the amino acid side chain, at the amino and/or at the carboxy terminus. They encompass for example acetylations, acylations, ADP ribosylations, amidations, covalent linkages to flavins, haeme moieties, nucleotides or nucleotide derivatives, lipids or lipid derivatives or phosphatidylinositol, cyclizations, the formation of disulphide bridges, demethylations, the formation of cystine, formylations, gamma-carboxylations, glycosylations, hydroxylations iodinations, methylations, myristoylations, oxidations, proteolytic processings, phosphorylations, selenoylations and tRNA-mediated additions of amino acids.

The polypeptides according to the invention may exist in the form of “mature” proteins or as parts of larger proteins, for example as fusion proteins. They may furthermore have secretion or leader sequences, pro-sequences, sequences which allow simple purification, such as repeated histidine residues, or additional stabilizing amino acids.

The polypeptides according to the invention, in particular the polypeptide in accordance with SEQ ID NO:2, need not constitute complete plant phosphomevalonate kinases, but may also just be fragments of these as long as they retain at least the biological activity of the complete plant phosphomevalonate kinase. Polypeptides which exert the same type of biological activity as a phosphomevalonate kinase with an amino acid sequence in accordance with SEQ ID NO:2 are still considered to be in accordance with the invention. The polypeptides according to the invention in this case need not be able to be derived from Arabidopsis phosphomevalonate kinases. Polypeptides which correspond to phosphomevalonate kinases of, for example, the plants stated hereinbelow, or to fragments thereof, and which are still capable of exerting the biological activity thereof are still considered to be in accordance with the invention: tobacco, maize, wheat, barley, oats, rice, rye, tomatoes, legumes, potato plants, Lactuca sativa, other Brassicaceae, woody species, Physcomitrella patens.

Compared with the corresponding region of naturally occurring phosphomevalonate kinases, the polypeptides according to the invention may exhibit deletions or amino acid substitutions as long as they still exert at least the biological activity of the complete kinase. Conservative substitutions are preferred. Such conservative substitutions encompass variations, where one amino acid is replaced by another amino acid from among the following group:

1. Small aliphatic residues, nonpolar residues or residues of low polarity: Ala, Ser, Thr, Pro and Gly;

2. Polar, negatively charged residues and their amides: Asp, Asn, Glu and Gln;

3. Polar, positively charged residues: His, Arg and Lys;

4. Large aliphatic nonpolar residues: Met, Leu, Ile, Val und Cys; and

5. Aromatic residues: Phe, Tyr and Trp.

Preferred conservative substitutions can be seen from the following list:

Subject-matter of the present invention are thus also polypeptides which exert at least the biochemical reaction of the formation of 5-pyrophosphomevalonate from 5-phosphomevalonate, such as phosphomevalonate kinase, and which encompass an amino acid sequence which exhibits at least 60% identity, preferably 80% identity, especially preferably 90% identity, very especially preferably 97-99% identity, with the sequence in accordance with SEQ ID NO:2 over its entire length.

The degree of identity of the amino acid sequences is preferably determined with the aid of the program BLASTP+BEAUTY Version 2.0.14. (Altschul et al., 1997).

A preferred embodiment of the polypeptides according to the invention is the phosphomevelonate kinase (PMVK) with the amino acid sequence in accordance with SEQ ID NO:2.

The PMVK amino acid sequence has a potential ATP binding site in the region of amino acids 177 to 186, which is typical of kinases.

The term “biological activity of a phosphomevalonate kinase” as used in the present context refers to the ability to convert 5-phosphomevalonate into 5-pyro-phosphomevalonate with consumption of ATP and formation of ADP.

The nucleic acids according to the invention can be prepared in the customary manner. For example, the nucleic acid molecules can be synthesized chemically in their entirety. It is also possible chemically to synthesize short sections of the nucleic acids according to the invention, and such oligonucleotides can be radiolabelled or labelled with a fluorescent dye. The labelled oligonucleotides can also be used for screening cDNA libraries generated starting from plant mRNA. Clones which hybridize with the labelled oligonucleotides are selected for isolating the DNA fragments in question. After characterization of the DNA which has been isolated, the nucleic acids according to the invention are obtained in a simple manner.

Alternatively, the nucleic acids according to the invention can be generated by means of PCR methods using chemically synthesized oligonucleotides.

The term “oligonucleotide(s)” as used in the present context denotes DNA molecules composed of 10 to 50 nucleotides, preferably 15 to 30 nucleotides. They are synthesized chemically and can be used as probes.

Subject-matter of the invention are also polypeptides with phosphomevalonate kinase activity which are encoded by an abovementioned DNA.

The skilled worker knows that the polypeptides of the present invention can be obtained by various routes, for example by chemical methods such as the solid-phase method. The use of recombinant methods is recommended for obtaining larger amounts of protein. Expression of a cloned phosphomevalonate kinase gene or fragments thereof can be effected in a series of suitable host cells with which the skilled worker is familiar. To this end, a phosphomevalonate kinase gene is introduced into a host cell with the aid of known methods.

The integration of the cloned phosphomevalonate kinase gene into the chromosome of the host cell is within the scope of the present invention. Preferably, the gene or fragments thereof are introduced into a plasmid and the coding regions of the phosphomevalonate kinase gene or fragments thereof are linked functionally with a constitutive or inducible promoter.

The basic steps for generating the recombinant phosphomevalonate kinase are:

1. Obtaining a natural, synthetic or semisynthetic DNA which encodes phosphomevalonate kinase.

2. Introduction of this DNA into an expression vector which is suitable for expressing phosphomevalonate kinase, either alone or as fusion protein.

3. Transformation of a suitable, preferably prokaryotic, host cell with this expression vector.

4. Growing this transformed host cell in a manner which is suitable for expressing phosphomevalonate kinase.

5. Cell harvest and purification of phosphomevalonate kinase by suitable known methods.

The coding region of phosphomevalonate kinase can be expressed in E. coli by the customary methods. Suitable expression systems for E. coli are commercially available, for example the expression vectors of the pET series, for example pET3a, pET23a, pET28a with His tag or pET32a with His tag for simple purification and thioredoxin fusion for increasing the solubility of the enzyme expressed, and pGEX with glutathione synthetase fusion, and the pSPORT vectors. The expression vectors are transformed into λ DE3 lysogenic E. coli strains, for example BL21(DE3), HMS 174(DE3) or AD494(DE3). After the growth of the cells under standard conditions which are familiar to the skilled worker, expression is induced by means of IPTG. After induction, the cells are incubated for 3 to 24 at temperatures from 18 to 37° C. The cells are disrupted by sonification in breaking buffer (10 to 200 mM sodium phosphate, 100 to 500 mM NaCl, pH 5 to 8). The protein expressed can be purified by chromatographic methods, in the case of protein expressed with His tag by chromatography on an Ni-NTA column.

Expression of the protein in commercially available yeast strains (for example Pichia pastoris ) or in insect cell cultures (for example Sf9 cells) constitutes another advantageous approach.

Alternatively, the proteins may also be expressed in plants.

Subject-matter of the present invention are also methods for finding chemical compounds which bind to the polypeptides according to the invention and modify their properties. Owing to the many functions of the terpenoids which make necessary the formation of the precursor isopentyl diphosphate and thus the function of the phosphomevalonate kinase according to the invention, modulators which affect the activity of the enzyme constitute new growth-regulatory or herbicidally active compounds. Modulators may take the form of agonists or antagonists, or activators or inhibitors.

Subject-matter of the present invention is therefore in particular also the use of plant phosphomevalonate kinases as targets for herbicides and their use in methods for finding modulators of this polypeptide. In such methods, the phosphomevalonate kinases can be employed directly, in extracts or in purified form, or they can be formed indirectly via expression of the DNA encoding them.

Subject-matter of the present invention is therefore also the use of nucleic acids encoding plant PMVK, DNA constructs comprising them, host cells comprising them, or of antibodies binding to PMVK for finding PMVK modulators.

The term “agonist” as used in the present context refers to a molecule which accelerates or increases the activity of phosphomevalonate kinase.

The term “antagonist” as used in the present context refers to a molecule which slows down or prevents the activity of phosphomevalonate kinase.

The term “modulator” as used in the present context constitutes the generic term for agonist or antagonist. Modulators can be small organochemical molecules, peptides or antibodies which bind to the polypeptides according to the invention. Other modulators may be small organochemical molecules, peptides or antibodies which bind to a molecule which, in turn, binds to the polypeptides according to the invention, thus affecting their biological activity. Modulators may constitute natural substrates and ligands or their structural or functional mimetics. The term “modulator”, however, does not encompass the natural substrates and ATP.

The modulators are preferably small organochemical compounds.

The binding of the modulators to the phosphomevalonate kinases according to the invention can alter the cellular processes in a manner which leads to the death of the plants treated therewith.

Subject-matter of the present invention are therefore also modulators, preferably inhibitors of the enzymatic activity of plant phosphomevalonate kinases, which were found with the aid of one of the processes described hereinbelow for identifying modulators of the phosphomevalonate kinase protein or a polypeptide which is homologous therewith.

Subject-matter of the invention is additionally the use of modulators of phosphomevalonate kinase as herbicides.

The present invention furthermore encompasses methods for finding chemical compounds which modify the expression of the polypeptides according to the invention. Such “expression modulators” too may constitute new growth-regulatory or herbicidal active compounds. Expression modulators can be small organochemical molecules, peptides or antibodies which bind to the regulatory regions of the nucleic acids encoding the polypeptides according to the invention. Furthermore, expression modulators can be small organochemical molecules, peptides or antibodies which bind to a molecule which, in turn, binds to regulatory regions of the nucleic acids encoding the polypeptides according to the invention, thus affecting their expression. Expression modulators may also be antisense molecules.

The present invention therefore also extends to the use of modulators of the polypeptides according to the invention or of expression modulators as plant growth regulators or herbicides.

Subject-matter of the present invention are also expression modulators of phosphomevalonate kinases which are formed with the aid of an above-described method of identifying expression modulators of the phosphomevalonate kinase protein or a polypeptide which is homologous thereto.

Subject-matter of the invention is also the use of expression modulators as herbicides.

Other methods according to the invention include high-throughput screening (HTS). Both host cells and cell-free preparations comprising the nucleic acids according to the invention and/or the polypeptides according to the invention may be used for this purpose.

In order to find modulators of the polypeptides according to the invention, a synthetic reaction mix (for example products of in vitro transcription) or a cellular component, such as a crude cell extract, or any other preparation comprising the polypeptide according to the invention can be incubated together with a labelled substrate or ligand of the polypeptides in the presence and absence of a candidate molecule, which may be an agonist or antagonist. The ability of the candidate molecule to increase or inhibit the activity of the polypeptide according to the invention can be seen from an increased or reduced binding of the labelled ligand or an increased or reduced conversion rate of the labelled substrate. Molecules which bind well and lead to an increased activity of the polypeptides according to the invention are agonists. Molecules which bind well but do not trigger the biological activity of the polypeptides according to the invention are probably good antagonists.

The detection of the biological activity of the polypeptides according to the invention can be improved by what is known as a reporter system. Reporter systems in this regard encompass, but are not limited to, colorimetrically labelled substrates which are converted into a product, or a reporter gene which responds to changes in the activity or the expression of the polypeptides according to the invention, or other known binding tests.

Modulators of the polypeptide according to the invention can also be found via enzymatic tests. The change in the enzyme activity by suitable modulators can either be measured directly or indirectly in a coupled enzyme test. The measurement can be carried out for example via the change in absorption owing to the decrease or increase of an optically active compound.

A further example of a method by means of which modulators of the polypeptides according to the invention can be found is a displacement test, in which the polypeptides according to the invention and a potential modulator are combined under conditions suitable for this test with a molecule which is known to bind to the polypeptides according to the invention, such as a natural substrate or ligand or a substrate or ligand mimetic. The polypeptides according to the invention themselves can be labelled, for example radiolabelled or colorimetrically labelled, so that the number of polypeptides which are bound to a ligand or which have undergone conversion can be determined accurately. In this manner, the efficacy of an agonist or antagonist can be assessed.

<160> NUMBER OF SEQ ID NOS: 5

<210> SEQ ID NO 1

<211> LENGTH: 2396

<212> TYPE: DNA

<213> ORGANISM: Arabidopsis thaliana

<220> FEATURE:

<221> NAME/KEY: CDS

<222> LOCATION: (685)..(2199)

<400> SEQUENCE: 1

gtcgacccac gcgtccgggc cgaccttctt cttcttcctt aagacaacac at

#aatgatag     60

aagcaaactg gggaagatga agatggagtg gtgaagaaca aaaccgtata ac

#cgttcggt    120

tcagaggtgc cgaaccgaac cgacccgtaa accgaaatcc tcaaaagaaa tt

#gccgatcg    180

gtttgctact gttcaaaacc tcggtgccga gaaccgaaac tgtcggtttt tt

#cggttcgg    240

gtttctcggt ttcttccgaa ctcccaggcc tagtttggtt ttatttttca cg

#agttttgc    300

ttctcttttc atcggcgacg acgacgtcga gtttctgtca aaacgttaac ga

#tccgactc    360

gagcgtcgac agtaagagaa gaagacagcg attgtgtgta gatcgacggc ga

#acgtgtgt    420

cgatccgtct cgatcgacgg agaatacgtt tcgatccggt ttcgatccaa at

#cggagagt    480

ttgaggatct aaatcggaaa ttgcattaat actcatctcc aatctcttct ga

#agagtccg    540

aatccgatct accaccacta ctcgtaccgc cggtcattta ctgccgccga tt

#tcaaatta    600

tccgatcatt tccggcgata tccaatcgca gactgaggtg aatctggggt tt

#tgatcagc    660

gattatcttt gtcactcttt gaaa atg gct gtt gtt gct t

#ct gct cct ggg       711

#         Met Ala Val Val Ala

#Ser Ala Pro Gly

#           1

#        5

aaa gtt ttg atg act gga ggc tac ctt gta ct

#c gag aag cca aat gca      759

Lys Val Leu Met Thr Gly Gly Tyr Leu Val Le

#u Glu Lys Pro Asn Ala

10

# 15

# 20

# 25

ggg ctt gtg ttg agt aca aat gca cgg ttt ta

#c gcg att gtg aag cca      807

Gly Leu Val Leu Ser Thr Asn Ala Arg Phe Ty

#r Ala Ile Val Lys Pro

30

#                 35

#                 40

atc aac gaa gaa gtc aag cct gaa agt tgg gc

#a tgg aaa tgg aca gat      855

Ile Asn Glu Glu Val Lys Pro Glu Ser Trp Al

#a Trp Lys Trp Thr Asp

45

#             50

#             55

gtc aaa tta aca tca cca cag ctc tcg aga ga

#a agc atg tat aaa ctg      903

Val Lys Leu Thr Ser Pro Gln Leu Ser Arg Gl

#u Ser Met Tyr Lys Leu

60

#         65

#         70

tca ctg aat cat ttg act ctt cag tct gtg tc

#t gca agt gat tca aga      951

Ser Leu Asn His Leu Thr Leu Gln Ser Val Se

#r Ala Ser Asp Ser Arg

75

#     80

#     85

aac ccc ttt gta gag cat gcg ata cag tat gc

#t ata gct gct gct cat      999

Asn Pro Phe Val Glu His Ala Ile Gln Tyr Al

#a Ile Ala Ala Ala His

90

# 95

#100

#105

ttg gca acc gag aag gac aaa gaa tca ttg ca

#c aaa ctc tta ttg caa     1047

Leu Ala Thr Glu Lys Asp Lys Glu Ser Leu Hi

#s Lys Leu Leu Leu Gln

110

#               115

#               120

ggt ctt gat ata aca ata tta ggc tcc aat ga

#c ttt tac tca tat cgg     1095

Gly Leu Asp Ile Thr Ile Leu Gly Ser Asn As

#p Phe Tyr Ser Tyr Arg

125

#           130

#           135

aac cag ata gaa tcg gct ggg ctt cca ttg ac

#a cca gaa tcg ctg ggt     1143

Asn Gln Ile Glu Ser Ala Gly Leu Pro Leu Th

#r Pro Glu Ser Leu Gly

140

#       145

#       150

acc ctt gca ccg ttt gca tca atc aca ttc aa

#t gct gcg gag tca aat     1191

Thr Leu Ala Pro Phe Ala Ser Ile Thr Phe As

#n Ala Ala Glu Ser Asn

155

#   160

#   165

ggt gct aat tcc aag cct gaa gta gca aaa ac

#t ggc tta ggt tct tct     1239

Gly Ala Asn Ser Lys Pro Glu Val Ala Lys Th

#r Gly Leu Gly Ser Ser

170                 1

#75                 1

#80                 1

#85

gca gca atg aca aca gct gtg gtt gca gct ct

#g tta cat tat ctt gga     1287

Ala Ala Met Thr Thr Ala Val Val Ala Ala Le

#u Leu His Tyr Leu Gly

190

#               195

#               200

gtg gtt gac cta tct gat cca tgt aaa gaa gg

#a aag ttt ggc tgt tct     1335

Val Val Asp Leu Ser Asp Pro Cys Lys Glu Gl

#y Lys Phe Gly Cys Ser

205

#           210

#           215

gat cta gat gtt atc cat atg ata gca caa ac

#g tct cat tgt ctt gca     1383

Asp Leu Asp Val Ile His Met Ile Ala Gln Th

#r Ser His Cys Leu Ala

220

#       225

#       230

caa ggg aag gtc gga agt ggg ttt gat gtc ag

#c tgt gct gtc tat gga     1431

Gln Gly Lys Val Gly Ser Gly Phe Asp Val Se

#r Cys Ala Val Tyr Gly

235

#   240

#   245

agt cag cgt tat gtt cgc ttc tct cca gaa gt

#c ttg tca ttt gct cag     1479

Ser Gln Arg Tyr Val Arg Phe Ser Pro Glu Va

#l Leu Ser Phe Ala Gln

250                 2

#55                 2

#60                 2

#65

gtt gca gta aca ggt ctg cca tta aat gaa gt

#t att ggt aca att ttg     1527

Val Ala Val Thr Gly Leu Pro Leu Asn Glu Va

#l Ile Gly Thr Ile Leu

270

#               275

#               280

aag gga aaa tgg gac aat aag aga act gag tt

#c tct tta cca cca ctg     1575

Lys Gly Lys Trp Asp Asn Lys Arg Thr Glu Ph

#e Ser Leu Pro Pro Leu

285

#           290

#           295

atg aat ctt ttc ctt gga gaa cct gga agt gg

#t gga tcc tcc aca cca     1623

Met Asn Leu Phe Leu Gly Glu Pro Gly Ser Gl

#y Gly Ser Ser Thr Pro

300

#       305

#       310

tca atg gta ggt gca gta aag aag tgg caa at

#g tct gat cca gag aag     1671

Ser Met Val Gly Ala Val Lys Lys Trp Gln Me

#t Ser Asp Pro Glu Lys

315

#   320

#   325

gca cga gaa aac tgg cag aat ttg tca gat gc

#a aat tta gaa ctg gaa     1719

Ala Arg Glu Asn Trp Gln Asn Leu Ser Asp Al

#a Asn Leu Glu Leu Glu

330                 3

#35                 3

#40                 3

#45

act aag cta aac gat ctg agc aaa tta gct aa

#a gac cac tgg gat gtt     1767

Thr Lys Leu Asn Asp Leu Ser Lys Leu Ala Ly

#s Asp His Trp Asp Val

350

#               355

#               360

tat cta cga gtc att aag tct tgt agt gtg ct

#t act tct gaa aag tgg     1815

Tyr Leu Arg Val Ile Lys Ser Cys Ser Val Le

#u Thr Ser Glu Lys Trp

365

#           370

#           375

gtg tta cat gct act gaa cca atc aac gaa gc

#c att att aaa gaa ctc     1863

Val Leu His Ala Thr Glu Pro Ile Asn Glu Al

#a Ile Ile Lys Glu Leu

380

#       385

#       390

tta gag gca aga gaa gct atg ttg agg atc ag

#a att ctt atg cgt cag     1911

Leu Glu Ala Arg Glu Ala Met Leu Arg Ile Ar

#g Ile Leu Met Arg Gln

395

#   400

#   405

atg ggt gag gcg gct agc gtt ccg ata gag cc

#t gaa tct caa act caa     1959

Met Gly Glu Ala Ala Ser Val Pro Ile Glu Pr

#o Glu Ser Gln Thr Gln

410                 4

#15                 4

#20                 4

#25

ctt ttg gat tct aca atg agt gct gaa gga gt

#t cta ctt gct ggt gtt     2007

Leu Leu Asp Ser Thr Met Ser Ala Glu Gly Va

#l Leu Leu Ala Gly Val

430

#               435

#               440

cct gga gct ggt gga ttt gat gcc ata ttt gc

#a atc act tta ggg gat     2055

Pro Gly Ala Gly Gly Phe Asp Ala Ile Phe Al

#a Ile Thr Leu Gly Asp

445

#           450

#           455

tcc ggc acc aaa ctg acc cag gca tgg agt tc

#g cac aat gtt ttg gcc     2103

Ser Gly Thr Lys Leu Thr Gln Ala Trp Ser Se

#r His Asn Val Leu Ala

460

#       465

#       470

ttg ttg gtg aga gaa gat cca cat ggc gtt tg

#c cta gaa agt ggt gat     2151

Leu Leu Val Arg Glu Asp Pro His Gly Val Cy

#s Leu Glu Ser Gly Asp

475

#   480

#   485

cca cga acc aca tgt att act tca ggc gtt tc

#a tca att cac ctt gag     2199

Pro Arg Thr Thr Cys Ile Thr Ser Gly Val Se

#r Ser Ile His Leu Glu

490                 4

#95                 5

#00                 5

#05

taaacaacat tgtttcagtg tccaattatt aggtgcgtca ccaagttcgg tt

#gagtatac   2259

tgttttgcat atagacttgg gtgctaaatt tcttggtgta agcattttta ta

#cccattgt   2319

aaggtcttta actcttggaa aacttgcggg aaaataaaat aaagttgatt tc

#aaatcttc   2379

tcaaaaaaaa aaaaaaa

#

#

# 2396

<210> SEQ ID NO 2

<211> LENGTH: 505

<212> TYPE: PRT

<213> ORGANISM: Arabidopsis thaliana

<400> SEQUENCE: 2

Met Ala Val Val Ala Ser Ala Pro Gly Lys Va

#l Leu Met Thr Gly Gly

1               5

#                 10

#                 15

Tyr Leu Val Leu Glu Lys Pro Asn Ala Gly Le

#u Val Leu Ser Thr Asn

20

#             25

#             30

Ala Arg Phe Tyr Ala Ile Val Lys Pro Ile As

#n Glu Glu Val Lys Pro

35

#         40

#         45

Glu Ser Trp Ala Trp Lys Trp Thr Asp Val Ly

#s Leu Thr Ser Pro Gln

50

#     55

#     60

Leu Ser Arg Glu Ser Met Tyr Lys Leu Ser Le

#u Asn His Leu Thr Leu

65

# 70

# 75

# 80

Gln Ser Val Ser Ala Ser Asp Ser Arg Asn Pr

#o Phe Val Glu His Ala

85

#                 90

#                 95

Ile Gln Tyr Ala Ile Ala Ala Ala His Leu Al

#a Thr Glu Lys Asp Lys

100

#           105

#           110

Glu Ser Leu His Lys Leu Leu Leu Gln Gly Le

#u Asp Ile Thr Ile Leu

115

#       120

#       125

Gly Ser Asn Asp Phe Tyr Ser Tyr Arg Asn Gl

#n Ile Glu Ser Ala Gly

130

#   135

#   140

Leu Pro Leu Thr Pro Glu Ser Leu Gly Thr Le

#u Ala Pro Phe Ala Ser

145                 1

#50                 1

#55                 1

#60

Ile Thr Phe Asn Ala Ala Glu Ser Asn Gly Al

#a Asn Ser Lys Pro Glu

165

#               170

#               175

Val Ala Lys Thr Gly Leu Gly Ser Ser Ala Al

#a Met Thr Thr Ala Val

180

#           185

#           190

Val Ala Ala Leu Leu His Tyr Leu Gly Val Va

#l Asp Leu Ser Asp Pro

195

#       200

#       205

Cys Lys Glu Gly Lys Phe Gly Cys Ser Asp Le

#u Asp Val Ile His Met

210

#   215

#   220

Ile Ala Gln Thr Ser His Cys Leu Ala Gln Gl

#y Lys Val Gly Ser Gly

225                 2

#30                 2

#35                 2

#40

Phe Asp Val Ser Cys Ala Val Tyr Gly Ser Gl

#n Arg Tyr Val Arg Phe

245

#               250

#               255

Ser Pro Glu Val Leu Ser Phe Ala Gln Val Al

#a Val Thr Gly Leu Pro

260

#           265

#           270

Leu Asn Glu Val Ile Gly Thr Ile Leu Lys Gl

#y Lys Trp Asp Asn Lys

275

#       280

#       285

Arg Thr Glu Phe Ser Leu Pro Pro Leu Met As

#n Leu Phe Leu Gly Glu

290

#   295

#   300

Pro Gly Ser Gly Gly Ser Ser Thr Pro Ser Me

#t Val Gly Ala Val Lys

305                 3

#10                 3

#15                 3

#20

Lys Trp Gln Met Ser Asp Pro Glu Lys Ala Ar

#g Glu Asn Trp Gln Asn

325

#               330

#               335

Leu Ser Asp Ala Asn Leu Glu Leu Glu Thr Ly

#s Leu Asn Asp Leu Ser

340

#           345

#           350

Lys Leu Ala Lys Asp His Trp Asp Val Tyr Le

#u Arg Val Ile Lys Ser

355

#       360

#       365

Cys Ser Val Leu Thr Ser Glu Lys Trp Val Le

#u His Ala Thr Glu Pro

370

#   375

#   380

Ile Asn Glu Ala Ile Ile Lys Glu Leu Leu Gl

#u Ala Arg Glu Ala Met

385                 3

#90                 3

#95                 4

#00

Leu Arg Ile Arg Ile Leu Met Arg Gln Met Gl

#y Glu Ala Ala Ser Val

405

#               410

#               415

Pro Ile Glu Pro Glu Ser Gln Thr Gln Leu Le

#u Asp Ser Thr Met Ser

420

#           425

#           430

Ala Glu Gly Val Leu Leu Ala Gly Val Pro Gl

#y Ala Gly Gly Phe Asp

435

#       440

#       445

Ala Ile Phe Ala Ile Thr Leu Gly Asp Ser Gl

#y Thr Lys Leu Thr Gln

450

#   455

#   460

Ala Trp Ser Ser His Asn Val Leu Ala Leu Le

#u Val Arg Glu Asp Pro

465                 4

#70                 4

#75                 4

#80

His Gly Val Cys Leu Glu Ser Gly Asp Pro Ar

#g Thr Thr Cys Ile Thr

485

#               490

#               495

Ser Gly Val Ser Ser Ile His Leu Glu

500

#           505

<210> SEQ ID NO 3

<211> LENGTH: 611

<212> TYPE: DNA

<213> ORGANISM: Medicago truncatula

<400> SEQUENCE: 3

ctgttatctg agttgaagaa atatcacaat atcaatggcc gtggtggttg ct

#tctgctcc     60

tgggaaggtg ttaatgaccg gtggctacct agttttagag agacctaatg ct

#ggacttgt    120

tcttagtact aatgctcgtt tttatgctat tgtcaaacca atctatcctc aa

#actaaacc    180

tgattcttgg gcttgggctt ggtcagatgt cagattaaca tctcctcaac tc

#tccagaga    240

agccttctat aaattagcac tcaaaaatct taccatccaa actgtttcct ca

#agtgaaac    300

aaggaaccct tttgtggaat atgctgtgca atactccgtg gctgccgcct at

#gcaacagc    360

tgaccagaat aaaaaggact tgttgcacaa actacttttg caaggtcttg ac

#attacaat    420

tttgggttcc aatgattttt attcttatag gaatgagatt gagagacacg ga

#ctcccttt    480

gacatcagaa tcattggcca cccttccgcc ttttgcctcc atttctttca at

#actgatga    540

tgctaatgga aggaattgta agcctgaaat tgccaaaact ggtttgggct ca

#tctgcagc    600

aatgacaacc g

#

#

#      611

<210> SEQ ID NO 4

<211> LENGTH: 728

<212> TYPE: DNA

<213> ORGANISM: Gossypium hirsutum

<220> FEATURE:

<221> NAME/KEY: unsure

<222> LOCATION: 715

<223> OTHER INFORMATION: n can be any nucleotid

#e

<400> SEQUENCE: 4

cgtttttacg ctattgttaa gccaattcat gaagctatca agcctgaaag ct

#gggcatgg     60

tcttggaccg atgtcaagct aacatctcct cagctttcca gagaaagcat gt

#ataaattg    120

tctcggaaac atttaacact tcagtgtgta tcttcaagtg aatcaaggaa cc

#cttttgta    180

gaaaatgcta ttcaatatac tatagcagct gcacatgcaa catttgacaa ga

#ataagaaa    240

gaggcattag ataaactact cttacaaggt cttgatatta cgatcttagg tt

#gcaatgac    300

ttttactcat acaggaatca gatagaagca cttggtcttc cgttgacacc tg

#aagcattg    360

gctactctac caccgtttac atcaattaca ttcaattctg aggaatcaaa tg

#gagcaaat    420

tgcaaacctg aagttgcaaa aactggattg ggttcatctg cagcaatgac aa

#ctgctgta    480

gttgctgctt tacttcatta tcttggtgtt gttaaccttt ccacctcttc tg

#cagatcaa    540

caccaagaaa ataagaattc cacagatctc gatattgtgc atatgatagc tc

#aaagtgcc    600

cactgtattg cccaaggtaa agttggcagt ggctttgatg tcagttctgc tg

#tctatggg    660

agtcagcgtt atgttcgttt ttcaccaaaa gtgctttctg ctgctcaggc tg

#cantgaaa    720

gggatgcc

#

#

#         728

<210> SEQ ID NO 5

<211> LENGTH: 571

<212> TYPE: DNA

<213> ORGANISM: Pinus radiata

<400> SEQUENCE: 5

cacaggcgaa accctctcct gctgctcacg gttgataaac cctcaatatt tg

#cggtaggg     60

ctccagattt actgcaatct gccagtaaga gtccgttgtg gcggaagaga gc

#tgccgaga    120

gctgccgagc tggagagcac cattcgcacc atatagagaa gggggttgat ag

#attcctgg    180

tcaaggaaaa ctgacaataa ggtgaaaaaa acaataatta ccttcagatt at

#ctgatcat    240

cacatggctg tagttgtgtc agctcctggt aaggttttaa taacaggagc tt

#atctaatt    300

cttgagaagc caaatccagg acttgtgctt accaccacag ctcgcttcta cg

#ccattgtg    360

aagccactgc ggactagcac agattccagt agttgggcat ggctatggac ag

#atgtgaaa    420

ttaacatcgc ctcagcttgc aaaggaggcc atctacaagc tatctctgaa ga

#ctcttagc    480

ctgcaaaatg ttgcttcttc aagtagcaat ggtaatcctt ttgtggaaca ag

#cagtgcaa    540

tttgctgttg cagctgcaaa agaagccttt g

#

#         571