Arabidopsis MPC1 gene and methods for controlling flowering time

The present invention relates to genes for floral regulation of plants and to methods for controlling plant flowering by regulating the expression of said gene. The present invention also relates to transgenic plants whose flowering time is modified in comparison with wild type plants by regulating the expression of said gene and to methods for generating said transgenic plants.

In order to resolve the worldwide food problem, developing technology for increasing the yield of food using biotechnology has been desired. Grain, which is one of main crops, is seed of plants and some vegetables are fruit of plants. For productivity increase of these plants, floral regulation for controlling growth of plants is an important key technology. On the other hand, flowering inhibition of vegetables, whose vegetative organs such as leaves or roots are marketed, prevents vegetative organs from stopping their growth and often increases their productivity. In addition, for many crops the suitable cultivating places are limited because of their species specificity of hereditary flowering behavior depending on environment. Modification of these properties by flowering regulation can expand the suitable cultivating places.

In molecular genetic studies using model plants such as Arabidopsis thaliana and Antirrhinum majas, many genes involved in identity determination of floral meristems or morphogenesis of floral organs have been isolated. Among these genes LEAFY and APETALA-1 genes are known to be forcedly expressed in the host plant Arabidopsis or poplar when introduced into these plants, thereby flowering the plants earlier. Since these genes are not fundamentally involved in floral budding (the transition from vegetative growth to reproductive growth), the use of these genes alone cannot arbitrarily regulate flowering. If the function of these genes is inhibited, the shape of inflorescence is changed, which is obvious from the phenotype of the mutants, and flowering cannot be regulated.

The embryonic flower mutant of Arabidopsis, in which flowering occurs immediately after germination, is known (Sung et al. (1992), Science, vol.258: p1645-). In this mutant, the function of a gene that maintains vegetative growth for a certain period of time after germination is thought to be lost. The flowering of wild-type Arabidopsis is thought to be inhibited by the expression of this gene. Although the approximate location of this gene on the chromosome is reported (Yang et al. (1995), Dev. Biol., vol.169: p421-), the result is far from helping the isolation of the gene and the gene has not yet been isolated.

An objective of the present invention is to isolate a gene for floral regulation (flowering regulating gene) and to provide a transgenic plant into which the gene is introduced. If a fundamental gene that regulates flowering is isolated, flowering time can be freely controlled by artificially regulating this gene.

The present inventors have succeeded in isolating mutant Arabidopsis that exhibits flowering immediately after germination because the function of the flowering regulating gene is lost and in identifying a single gene, which was mutated, in a wide region of the chromosome and isolating it. Furthermore, the present inventors have confirmed that this gene has flowering inhibiting function by introducing the gene into Arabidopsis and expressing it. Based on these findings, the present inventors have completed the present invention.

Moreover, the present inventors have discovered that the flowering regulating gene isolated from any kind of plant by hybridization or PCR technique based on the sequence of Arabidopsis flowering regulating gene has the function that complements the mutation of the Arabidopsis super early flowering mutant, inhibits flowering, and induces normal differentiation of stems and leaves.

Thus, the present invention relates to novel flowering regulating genes that exist extensively in plants, proteins with flowering regulating activity encoded by said genes, transgenic plants in which the expression of said gene is modified, methods for generating these plants, and methods for controlling the flowering time of plants by regulating the expression of said genes. More specifically, the present invention relates to

(1) a DNA encoding a protein having flowering regulating activity, wherein said DNA selected from the group consisting of:

i) a DNA encoding a protein comprising the amino acid sequence of SEQ ID NO: 1;

ii) a DNA encoding a protein comprising the amino acid sequence substantially identical to that of SEQ ID NO: 1;

iii) a DNA hybridizing a DNA encoding the protein comprising the amino acid sequence of SEQ ID NO:1;

iv) a DNA encoding a protein comprising amino acid sequences showing 50% or more and 60% or more homology with amino acids 278 to 348 and 465 to 607, respectively, of the amino acid sequence of SEQ ID NO: 1;

v) a DNA encoding a protein comprising the amino acid sequence of SEQ ID NO: 8.

vi) a DNA encoding a protein comprising the amino acid sequence substantially identical to that of SEQ ID NO: 8.

vii) a DNA hybridizing a DNA encoding the protein comprising the amino acid sequence of SEQ ID NO: 8; and

viii) a DNA encoding a protein comprising amino acid sequences showing 50% or more and 60% or more homology with amino acids 282 to 352 and 450 to 592, respectively, of the amino acid sequence of SEQ ID NO: 8.a DNA encoding a protein having flowering regulating activity, wherein said protein comprises the amino acid sequence of SEQ ID NO: 1;

(2) the DNA of (1), wherein said DNA of i) comprises the coding region of the nucleotide sequence of SEQ ID NO: 2;

(3) the DNA of (1), wherein said DNA of v) comprises the coding region of the nucleotide sequence of SEQ ID NO: 9;

(4) the DNA of (1), encoding a protein having a zinc finger structure;

(5) a protein having flowering regulating activity, encoded by the DNA of (1);

(6) the protein of (5), comprising the amino acid sequence of SEQ ID NO: 1 or 8;

(7) a recombinant double-stranded DNA molecule comprising an expression cassette comprising the DNA of (1);

(8) a recombinant double-stranded DNA molecule comprising an expression cassette comprising the following constituent elements of i) to iii),

i) a promoter that can transcribe in plant cells,

ii) the DNA of (1) or a part of it fused to said promoter in sense or antisense direction, and selectively, and

iii) a signal involved in transcription termination of RNA molecules and polyadenylation, wherein the signal functions in plants;

(9) a transformant into which the recombinant double-stranded DNA molecule of (7) is introduced;

(10) a transgenic plant cell into which the recombinant double-stranded DNA molecule of (8) is introduced;

(11) a method for producing a protein of (5), wherein the method comprises

(a) cultivating a transformant of (9) and

(b) recovering a recombinant protein from said transformant or the culture supernatant of it;

(12) a transgenic plant comprising transgenic plant cells of (10);

(13) a method for producing a transgenic plant of (12), wherein said method comprises

(a) introducing the recombinant double-stranded DNA molecule of (8) into plant cells and

(b) regenerating said plant cells;

(14) a DNA encoding an antisense RNA complementary to a transcription product of a DNA of (1);

(15) a method for regulating the flowering time of a plant, wherein said method comprises introducing the whole or a part of a DNA of (1) or the whole or a part of a DNA of (14) into a plant and expressing it, thereby changing the activity of a flowering regulating protein; and

(16) an antibody that binds to a protein of (5).

The drawing shows the location of the DNA clones of the chromosome region containing Arabidopsis “MPC1” gene and the markers 16EB53 and z11-1. In the drawing the unfilled arrow shows the position and direction of “MPC1” gene and 11K22, 22K2, 19A20, and 20I12 show DNA clones.

“An expression cassette” used herein means a DNA molecule comprising a gene and constituent elements essential for the expression of the gene. Typically, it is a DNA molecule comprising (i) a promoter to express a structural gene in a host, (ii) the structural gene, and, if necessary, (iii) a terminator. The promoter varies depending on the host. For example, in order to produce a recombinant protein in a microorganism, a promoter functioning in the microorganism is used. For generating a transgenic plant, a promoter functioning in plant cells is used. An example of “a recombinant double-stranded DNA molecule comprising an expression cassette” is typically a vector comprising an expression cassette.

The present invention provides novel proteins regulating the flowering of plants and DNAs encoding said proteins. The nucleotide sequences of the cDNA and the genomic DNA of Arabidopsis-derived “MPC1”, which has been isolated by the present inventors, are shown in SEQ ID NO: 2 and 3, respectively. The amino acid sequence of Arabidopsis-derived “MPC1” protein encoded by the cDNA or genomic DNA is shown in SEQ ID NO: 1. The nucleotide sequences of the cDNA and the genomic DNA of rice-derived “Os-MPC1”, which has been isolated by the present inventors, are shown in SEQ ID NO: 9 and 10, respectively. The amino acid sequence of rice-derived “Os-MPC1” protein encoded by the cDNA or genomic DNA is shown in SEQ ID NO: 8.

The mutation of Arabidopsis-derived “MPC1” gene eliminates normal flowering regulating ability of plants and leads plant to flowering immediately after germination (super early flowering mutation). The present inventors have found that guanine is substituted with adenine at the nucleotide 5039 of “MPC1” genomic DNA (SEQ ID NO: 3) in “mpc1” mutant plants (Example 1). Since the C-terminal amino acid residues from 541 of “MPC1” protein are not translated by this base substitution, the deletion of the amino acid sequence after this mutation point is thought to diminish the normal flowering regulating function of “MPC1” protein. In other words, this deletion inhibits flowering function and leads plants to super early flowering. This phenomenon has been induced by introducing and expressing an antisense DNA in plants, thereby inhibiting “MPC1” protein expression (Example 2). Furthermore, the cDNA of rice-derived “Os-MPC1”, which shows significant homology with said Arabidopsis cDNA, also complement the super early flowering mutation of Arabidopsis (Example 5). It is therefore thought that these proteins exist widely in plants and regulate the flowering time.

Many plants including Arabidopsis vegetatively grow for a certain period of time after germination, flower, and reproductively grow. The genes of the present invention are essential to maintain vegetative growth, and to regulate the transition from vegetative growth to reproductive growth. In other words, the expression level of this gene regulates flowering. Therefore, the flowering time of plants can be changed by artificially regulating the expression of the genes of the present invention, which leads to productivity increase of useful plants.

DNAs used in this invention are not limited to DNAs encoding Arabidopsis-derived “MPC1” or rice-derived “Os-MPC1” protein mentioned above. Other DNAs encoding proteins functionally equivalent to these proteins can also be used.

An example of these DNAs is a DNA encoding a protein having an amino acid sequence substantially identical to that of Arabidopsis-derived “MPC1” or rice-derived “Os-MPC1” protein, whose amino acid sequence is shown in SEQ ID NO: 1 or 8. “An amino acid sequence substantially identical” used herein means a sequence in which changes such as deletion, substitution, addition, and/or insertion have occurred at one or more amino acid residues of the control amino acid sequence, and an amino acid sequence constitutes a protein having flowering regulating activity as the protein comprising the control amino acid sequence. Changes such as deletion, substitution, and addition can be performed at several amino acid residues, for example, by site-directed mutagenesis (Kunkel et al. (1985), Proc. Natl. Acad. Sci. USA, vol.82: p488-). Mutations of amino acids can also occur spontaneously.

Comparing amino acid sequences of proteins having flowering regulating activity of Arabidopsis and rice, high homology is found particularly in the region comprising zinc finger motifs and the region comprising an acidic amino acid cluster at the C-terminus. A zinc finger or zinc finger structure is a structure in which a part of a protein folds chelating zinc (Zn) to construct a protruding structure like a finger, and is thought to play an important role when the protein binds to nucleic acid or other protein (Roosenfeld et al. (1993), J. Biomol. Struct. Dyn., Vol.11:p557-). An amino acid sequence that can form a zinc finger structure is called a zinc finger motif, several types of which are known. Zinc fingers of Cys2-His2 (C2H2) type are found at the amino acids 306 to 327 of the Arabidopsis-derived “MPC1” protein and the amino acids 310 to 331 of rice-derived “Os-MPC1” protein. These motifs can be identified by, for example, a program such as “MOTIF” of “GenomeNet”, which is provided by Institute for Chemical Research, Kyoto University through the internet.

Acidic amino acid clusters are found in some kinds of transcription regulating proteins and sometimes play an important role in activating transcription (T. Tamura (1995), Mechanism of Transcriptional Regulation, Experimental Medicine Bioscience, Yodosha). These acidic amino acid clusters are found at amino acids 503 to 520 of Arabidopsis-derived “MPC1” protein and amino acids 488 to 505 of rice-derived “Os-MPC1” protein. These regions comprising a zinc finger motif or acidic amino acid cluster are likely to play an important role in flowering regulation of plants, and it is expected that high homology is kept in these regions of flowering regulation related proteins derived from plants other than Arabidopsis and rice.

The proteins having amino acid sequences substantially identical to that of Arabidopsis-derived “MPC1” or rice-derived “Os-MPC1” protein shown in SEQ ID NO: 1 or 8 are preferably those comprising amino acid sequences substantially identical to the regions of the above sequences comprising a zinc finger motif and a C-terminal acidic amino acid cluster.

A specific example thereof is a protein having flowering regulating activity, wherein the protein comprises amino acid sequences showing 50% or more and 60% or more homology with amino acids 278 to 348 and 465 to 607, respectively, of the amino acid sequence of SEQ ID NO: 1 (Arabidopsis), or a protein having flowering regulating activity, wherein the protein comprises amino acid sequences showing 50% or more and 60% or more homology with amino acids 282 to 352 and 450 to 592, respectively, of that of SEQ ID NO: 8 (rice).

Whether a protein has flowering regulating activity or not can be evaluated by, for example, introducing a DNA encoding said protein into super early flowering mutant plants. For example, a DNA encoding a test protein are introduced into super early flowering mutant plants such as “mpc1” Arabidopsis mutant, and expressed. The introduced DNA is judged to encode a protein having flowering regulating activity if it complements super early flowering mutant and differentiates normal stems and leaves as shown in Example 5. These DNA are thought to encode proteins having the same function as Arabidopsis-derived “MPC1” or rice-derived “Os-MPC1” protein (SEQ ID NO: 1 or 8, respectively).

In addition, other DNAs encoding proteins functionally equivalent to Arabidopsis-derived “MPC1” or rice-derived “Os-MPC1” protein can be screened by hybridization technique using the whole or a part of the DNA sequence encoding the amino acid sequence of SEQ ID NO: 1 or 8 as a probe (Southern (1975), J. Mol. Biol., vol.98: p503-; Sambrook et al. (1989), Molecular Cloning, Cold Spring Harbor Laboratory Press). Partial sequences of “MPC1” or “Os-MPC1” used as probes are at least fourteen or more nucleotide sequences. For example, GeneImage system (Amersham) can be used for hybridization. In accordance with the protocol attached to the product, test DNAs are incubated overnight with labeled probes, and those that hybridizes with the probes can be screened by washing at 50° C. with 6×SSC and 0.1% SDS. Alternatively, DNAs encoding proteins functionally equivalent to Arabidopsis-derived “MPC1” or rice-derived “Os-MPC1” protein can be isolated from other plants by PCR technique using oligonucleotides specifically hybridizing with the DNA encoding the amino acid sequence constituting “MPC1” or “Os-MPC1” protein as primers (K. Shimamoto & T. Sasaki (1995), Protocols of PCR Experiments for Plants, Cell Engineering SUPPLEMENT, Plant Cell Engineering Series 2, Shujunsha).

Flowering regulating proteins encoded by DNAs obtained by such hybridization or PCR technique are thought to have high homology with Arabidopsis-derived “MPC1” or rice-derived “Os-MPC1” protein. The term “high homology” means 45% or more, preferably 60% or more, more preferably 75% or more, still more preferably 90% or more, and most preferably 95% or more homology with at least one amino acid sequence of these proteins. The homology may possibly become 45% or less when plural amino acid residues of the amino acid sequence encoded by the isolated DNA are deleted, added, or replaced. Even in this case, the DNA can encode a protein having the region essential for the function of flowering regulating proteins and having the equivalent flowering regulating activity. As mentioned above, it is important for the protein to exhibit the flowering regulating function that high homology exists, in particular, in regions comprising a zinc finger motif region and a C-terminal acidic amino acid cluster region.

The homology between two or more genes in terms of the nucleotide sequences or the amino acid sequences of the proteins encoded by the genes can be determined using software for gene analysis, for example, DNASIS (Hitachi Software Engineering). In the software, the programs “Homology Plot,” which plots homology as two-dimensional image, and “Maximum Matching,” in which sequences are aligned considering gaps, are available for calculating homology between two genes (Needleman, S. B. et al. (1970), J. Mol. Biol., vol.48: p443-). The “Multialignment” program aligns three or more kinds of sequences to clarify the homologous regions (Waterman, M. S. (1986), Nucleic Acids Research, vol.14: 9095-).

Examples of plants from which the DNAs of the present invention are isolated by hybridization or PCR technique include corn, wheat, barley, rye, potato, tobacco, sugar beet, sugarcane, rape seed, soybean, sunflower, cotton, orange, grape, peach, pear, apple, Japanese apricot, tomato, Chinese cabbage, cabbage, Japanese radish, carrot, pumpkin, cucumber, melon, parsley, orchid, chrysanthemum, lily, saffron, pine, eucalyptus, acacia, poplar, Japanese cedar, Japanese cypress, bamboo, and yew, in addition to Arabidopsis and rice, but are not limited thereto. The present inventors have succeeded in isolating a flowering regulating gene encoding a protein substantially the same as Arabidopsis-derived “MPC1” or rice-derived “Os-MPC1” protein from sugar beet using hybridization or PCR technique mentioned above (Example 6).

Flowering regulating proteins of the present invention can be produced as recombinant proteins or natural proteins. Recombinant proteins can be expressed with, for example, the expression system using E. coli as a host, as fusion proteins to glutathione S-transferase (Smith, D. B. et al. (1988), Gene vol.67: p32-) or as fusion proteins with histidine-tag (Nakamura et al. (1998), Protocols of Protein Experiments for Plants, Cell Engineering SUPPLEMENT, Plant Cell Engineering Series 9, Shujunsha). The desired protein expressed as a fusion protein in E. coli is isloated by purifying the fusion protein by affinity chromatography with glutathione or metal ions as ligands and cutting out the desired protein by an appropriate protease treatment. Natural proteins can be produced by known methods for preparing proteins from plants (Nakamura et al. (1998), Protocols of Protein Experiments for Plants, Cell Engineering SUPPLEMENT, Plant Cell Engineering Series 9, Shujunsha).

Using recombinant or natural flowering regulating proteins prepared by the method mentioned above, polyclonal or monoclonal antibodies against them can be generated (Nakamura et al. (1998), Protocols of Protein Experiments for Plants, Cell Engineering SUPPLEMENT, Plant Cell Engineering Series 9, Shujunsha). Polyclonal antibodies can be generated by, for example, the method below. A laboratory animal such as a mouse is immunized with the prepared protein or its partial fragments mixed with appropriate adjuvant by intraperitoneal or subcutaneous injection. Additional immunization is then performed 2 to 10 times every one to four week, preferably every one or two week. After the fourth week, the blood is collected, serum is obtained to serve as antibody, and the antibody titer is measured by, for example, western blotting. The obtained antibody can be used in various experiments.

Monoclonal antibodies can be produced by fusing myeloma cells and the spleen cells obtained from the laboratory animal such as a mouse immunized by the method mentioned above and cloning the hybridoma producing the desired antibody. The hybridoma is cultivated in an appropriate medium to obtain the desired monoclonal antibody from the culture supernatant. A large amount of antibody can be obtained when hybridoma is cultivated in ascites. For example, hybridoma is transplanted into a nude mouse and allowed to grow. The monoclonal antibody produced in ascites of said animal is then collected.

Plant flowering regulation of the present invention can be performed by enhancing or inhibiting the expression of DNAs encoding the flowering regulating proteins mentioned above in target plants. Specifically, transgenic plants are generated by introducing said DNA or the antisense DNA against said DNA to the target plant. The DNA or the antisense DNA can be placed under the control of an appropriate inducible promoter to subtly regulate the degree of activation or inhibition of flowering and flowering time.

These DNAs can be expressed by introducing, into plant cells, a recombinant double-stranded DNA molecule comprising an expression cassette comprising (i) a promoter that is transcribed in plant cells, (ii) the whole or a part of the DNA encoding a flowering regulating protein of the present invention fused at the downstream of the promoter in sense or antisense direction, and if necessary, (iii) a terminator sequence fused at the downstream of the DNA, which comprises a polyadenylation site essential for stabilizing the transcript. “A part of the DNA encoding a flowering regulating protein” used herein means a part of the DNA encoding a complete flowering regulating protein that regulates flowering when it is expressed in plant cells. The present invention includes these recombinant double-stranded DNA molecules. The recombinant double-stranded DNA molecules can have DNA sequences essential to transfer the molecule to host plant cells or to maintain it in the host cells at its 5′- and/or 3′-end as well as constituent elements described above.

An expression cassette can comprise a promoter to express constitutively or inducibly the DNA encoding the inserted flowering regulating protein of the present invention. Examples of promoters for constitutive expression are 35 S promoter of cauliflower mosaic virus (Odell et al. (1985), Nature, vol.313: p810-) and rice actin promoter (Zhang et al. (1991), Plant Cell, vol.3: p1155-). Examples of promoters for inducible expression are promoters known to express by external factors such as infection or invasion of fungi, bacteria, or virus, low or high temperature, dryness, irradiation of ultraviolet rays, contacting with specific compounds. Examples of these promoters are rice chitinase gene promoter (Xu et al. (1996), Plant Mol. Biol., vol.30: p387-) and tobacco PR protein gene promoter (Ohshima et al. (1990), Plant Cell, vol2: p95-), both of which are induced by infection or invasion of fungi, bacteria, or virus, rice “lip19” gene promoter that is induced by low temperature (Aguan et al. (1993), Mol. Gen. Genet., vol.240: p1-), Arabidopsis “HSP18.2” gene promoter that is induced by high temperature (Yoshida et al. (1995), Appl. Microbiol. Biotechnol., vol.44(3-4): p466-), rice “rab” gene promoter that is induced by dryness (Yamaguchi-Shinozaki et al. (1990), Plant Mol. Biol., vol.14(1): p29-), parsley chalcone synthase gene promoter that is induced by ultraviolet rays (Schulze-Lefert et al. (1989), EMBO J., vol.8: p651-), and corn alcohol dehydrogenase gene promoter that is induced under anaerobic conditions (Walker et al. (1987), Proc. Natl. Acad. Sci. USA vol.84: p6624-). Besides, rice chitinase gene promoter and tobacco PR protein gene promoter are induced by specific compound such as salicylic acid, and rice “rab” gene promoter by sprinkling of a plant hormone abscisic acid.

Various cloning vectors comprising the replication origin of E. coli and a marker gene for screening transformed bacterial cells are available to introduce the recombinant DNA molecules into plants. Examples of these vectors include pBR322, pUC series, and M13mp series. A desired sequence can be introduced into a vector at an appropriate restriction enzyme site. A plasmid DNA obtained can be characterized by restriction endonuclease cleavage site analysis, gel electrophoresis, and other biochemical-molecular biological methods. Once the plasmid DNA is prepared, it can be cleaved and ligated with another DNA. The sequence of the plasmid DNA can be cloned into the same plasmid or other plasmids.

When the whole of a DNA encoding a flowering regulating protein of the present invention, for example, the whole region of Arabidopsis-derived “MPC1” cDNA, shown in SEQ ID NO: 2, is ligated at the downstream of a promoter in sense direction, expression of the flowering regulating gene can be expressed constitutively or inducibly depending on the property of the promoter used. Then, the activity of the flowering regulating protein in plant cells constitutively or inducibly increases, and consequently, delay or inhibition of flowering can constitutively or inducibly is caused in plants.

When the whole or a part of a DNA encoding a flowering regulating protein, for example, the whole or a part region of Arabidopsis-derived “MPC1” cDNA, shown in SEQ ID NO: 2, is ligated at the downstream of a promoter mentioned above in antisense direction, the antisense RNA complementary to the transcript of “MPC1” cDNA can be constitutively or inducibly expressed depending on the property of the promoter used. The expression of a flowering regulating protein of the present invention is constitutively or inducibly inhibited in plant cells, and consequently, flowering can be enhanced constitutively or inducibly in plants. Antisense DNAs used do not have to encode the antisense RNAs completely complementary to the transcript of endogenous flowering regulating protein gene as long as it can inhibit the expression of endogenous flowering regulating protein.

In plants, when a gene is ligated in sense direction at the downstream of a promoter causing constitutive and strong expression, the expression of both of the gene introduced and the corresponding endogenous gene is sometimes inhibited (Montgomery (1998), Trends Genet., 14, 255-). This phenomenon is called co-suppression. When a flowering regulating gene of the present invention, for example, the whole region of Arabidopsis-derived “MPC1” cDNA, shown in SEQ ID NO: 2, is ligated in sense direction at the downstream of 35 S promoter, the expression of the endogenous flowering regulating protein in plant cells can be inhibited by co-suppression to enhance flowering in plants.

Moreover, when a part of a DNA encoding a flowering regulating protein of the present invention, for example, a part region of “MPC1” cDNA, shown in SEQ ID NO: 2, is ligated at the downstream of a promoter mentioned above in sense direction, an incomplete flowering regulating protein can be constitutively or inducibly expressed depending on the property of the promoter used. The incomplete flowering regulating protein that constitutively or inducibly accumulates in cells can inhibit the normal function of the flowering regulating protein, thereby enhancing flowering constitutively or inducibly in plants.

Both dicotyledon and monocotyledon can be used at target plants for generating plants whose flowering behavior is changed in comparison with wild type plants. Particularly important plants are grain (for example, rye, wheat, corn, barley, and rice), fruits (for example, orange, grope, peach, pear, apple, and Japanese apricot), vegetables (for example, tomato, Chinese cabbage, cabbage, Japanese radish, carrot, pumpkin, potato, cucumber, melon, and parsley), ornamental plants (for example, orchid, chrysanthemum, lily, and saffron), other industrial crops of economical importance (for example, tobacco, sugar beet, rape seed, soybean, sunflower, and cotton), and trees that require a long period till flowering (for example, eucalyptus, acacia, and poplar, which are used as wood pulp, and cedar, Japanese cypress, pine, bamboo, and yew, which are used as lumber).

Various methods can be used for introducing expression cassettes into plant host cells. Examples thereof are transformation of plants cells with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as a transformation mediator, direct introduction into protoplast (infection method, electroporation method, etc.), and particle gun method, but are not limited thereto.

Direct introduction into protoplast needs no special vectors. For example, simple plasmids such as pUC derivatives can be used. Some methods for introducing a desired gene into plant cells need other DNA sequences. For example, when a Ti or Ri plasmid is used to transform plant cells, at least right side sequence or usually both side sequences at the T-DNA region of Ti or Ri plasmid should be connected adjacent to a gene to be introduced.

When Agrobacterium is used to transform plant cells, an expression cassette to be introduced should be cloned in a special plasmid, an intermediate vector or binary vector. An intermediate vector is not replicated in Agrobacterium. An intermediate vector is transferred into Agrobacterium with a helper plasmid or by electroporation. Having regions homologous to T-DNA sequence, an intermediate vector is integrated into Ti or Ri plasmid of Agrobacterium by homologous recombination. Agrobacterium used as a host has to comprise vir region. Usually, Ti or Ri plasmid comprises vir region and can transfer T-DNA into plant cells by its function.

In contrast, since a binary vector can be replicated and maintained in Agrobacterium, if it is introduced into Agrobacterium with a helper plasmid or by electroporation, T-DNA on a binary vector can be transferred into plant cells by the function or vir region of the host. The present invention also includes intermediate vectors or binary vectors thus obtained, and microorganisms such as E. coli or Agrobacterium comprising them.

Transformed plant cells can be regenerated to a plant. The method for regeneration depends on the kind of the plant cells. Examples thereof are the methods of Fujimura et al. (Fujimura et al. (1995), Plant Tissue Culture Lett., vol.2: p74-) for rice, Shillito et al. (Shillito et al. (1989), Bio/Technology, vol.7: p581-) for corn, Visser et al. (Visser et al. (1989), Theor. Appl. Genet., vol.78: p594-) for potato, and Akama et al. (Akama et al. (1992), Plant Cell Rep., vol.12: p7-) for Arabidopsis. In plants generated by these methods or plants obtained from their vehicles for reproduction (for example, seeds, tubers, cuttings), the flowering regulating protein expression of the present invention changes in comparison with wild type plants, which changes the flowering behavior. The present invention includes transgenic plants thus obtained.

The present invention provides a novel gene that inhibits flowering of plants. When this gene is introduced into other plants and expressed in the plants, it can inhibits or enhance flowering of the plants.

Various cultivars of grain and vegetable that matures earlir or later than usual can be generated by regulating flowering, which produces such as agriculturally important value as expansion of the suitable cultivation place, increase of yield, and supply of crops with high value added. In particular, though the deterioration of quality by bolting and flowering is a problem in leaf and stem vegetables such as Chinese cabbage and root vegetables such as Japanese radish, the type of cultivation is limited at present. Therefore, flowering inhibition will bring a great effect such as the expansion of the cultivation season and the suitable cultivation place. Arbitrary flowering regulation will also be considerably useful if it is applied to a cultivar having superior characteristics such as good taste or strong disease resistance. Furthermore, enhancing the floral budding of fruits will increase their productivity or change the flowering time, which enables the production and shipment of fruits out of season. In addition, inhibiting flowering of wood will not only enhance alternation of generations by shortening the period required for flowering but also enhance vegetative growth or suppress allergy induction in humans caused by scatter of pollen, which is economically and socially significant.

The present invention is illustrated in detail below with reference to Examples, but is not to be construed as being limited thereto. Besides, methods for general gene recombination such as cleavage and ligation of DNAs, transformation of E. coli, determination of nucleotide sequences of genes, hybridization were performed, unless otherwise mentioned, based on manuals attached to commercial reagents and apparatus or laboratory books, for example, “Molecular Cloning” (Sambrook et al. (1989), Cold Spring Harbor Laboratory Press). In addition, cultivation of Arabidopsis using agar medium or soil, mating manipulation, preparation of genomic DNAs, genetic analysis are performed, unless otherwise mentioned, in accordance with laboratory books, for example, “Experimental Protocols for Model Plants” (Shimamoto & Okada (1996), Cell Engineering SUPPLEMENT, Plant Cell Engineering Series 4, Shujunsha).

<160> NUMBER OF SEQ ID NOS: 20

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<213> ORGANISM: Arabidopsis thaliana

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<221> NAME/KEY: ZN_FING

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Met Cys His Glu Asp Ser Arg Leu Arg Ile Ser Glu Glu Glu Glu Ile

1 5 10 15

Ala Ala Glu Glu Ser Leu Ala Ala Tyr Cys Lys Pro Val Glu Leu Tyr

20 25 30

Asn Ile Ile Gln Arg Arg Ala Ile Arg Asn Pro Leu Phe Leu Gln Arg

35 40 45

Cys Leu His Tyr Lys Ile Glu Ala Lys His Lys Arg Arg Ile Gln Met

50 55 60

Thr Val Phe Leu Ser Gly Ala Ile Asp Ala Gly Val Gln Thr Gln Lys

65 70 75 80

Leu Phe Pro Leu Tyr Ile Leu Leu Ala Arg Leu Val Ser Pro Lys Pro

85 90 95

Val Ala Glu Tyr Ser Ala Val Tyr Arg Phe Ser Arg Ala Cys Ile Leu

100 105 110

Thr Gly Gly Leu Gly Val Asp Gly Val Ser Gln Ala Gln Ala Asn Phe

115 120 125

Leu Leu Pro Asp Met Asn Arg Leu Ala Leu Glu Ala Lys Ser Gly Ser

130 135 140

Leu Ala Ile Leu Phe Ile Ser Phe Ala Gly Ala Gln Asn Ser Gln Phe

145 150 155 160

Gly Ile Asp Ser Gly Lys Ile His Ser Gly Asn Ile Gly Gly His Cys

165 170 175

Leu Trp Ser Lys Ile Pro Leu Gln Ser Leu Tyr Ala Ser Trp Gln Lys

180 185 190

Ser Pro Asn Met Asp Leu Gly Gln Arg Val Asp Thr Val Ser Leu Val

195 200 205

Glu Met Gln Pro Cys Phe Ile Lys Leu Lys Ser Met Ser Glu Glu Lys

210 215 220

Cys Val Ser Ile Gln Val Pro Ser Asn Pro Leu Thr Ser Ser Ser Pro

225 230 235 240

Gln Gln Val Gln Val Thr Ile Ser Ala Glu Glu Val Gly Ser Thr Glu

245 250 255

Lys Ser Pro Tyr Ser Ser Phe Ser Tyr Asn Asp Ile Ser Ser Ser Ser

260 265 270

Leu Leu Gln Ile Ile Arg Leu Arg Thr Gly Asn Val Val Phe Asn Tyr

275 280 285

Arg Tyr Tyr Asn Asn Lys Leu Gln Lys Thr Glu Val Thr Glu Asp Phe

290 295 300

Ser Cys Pro Phe Cys Leu Val Lys Cys Ala Ser Phe Lys Gly Leu Arg

305 310 315 320

Tyr His Leu Pro Ser Thr His Asp Leu Leu Asn Phe Glu Phe Trp Val

325 330 335

Thr Glu Glu Phe Gln Ala Val Asn Val Ser Leu Lys Thr Glu Thr Met

340 345 350

Ile Ser Lys Val Asn Glu Asp Asp Val Asp Pro Lys Gln Gln Thr Phe

355 360 365

Phe Phe Ser Ser Lys Lys Phe Arg Arg Arg Arg Gln Lys Ser Gln Val

370 375 380

Arg Ser Ser Arg Gln Gly Pro His Leu Gly Leu Gly Cys Glu Val Leu

385 390 395 400

Asp Lys Thr Asp Asp Ala His Ser Val Arg Ser Glu Lys Ser Arg Ile

405 410 415

Pro Pro Gly Lys His Tyr Glu Arg Ile Gly Gly Ala Glu Ser Gly Gln

420 425 430

Arg Val Pro Pro Gly Thr Ser Pro Ala Asp Val Gln Ser Cys Gly Asp

435 440 445

Pro Asp Tyr Val Gln Ser Ile Ala Gly Ser Thr Met Leu Gln Phe Ala

450 455 460

Lys Thr Arg Lys Ile Ser Ile Glu Arg Ser Asp Leu Arg Asn Arg Ser

465 470 475 480

Leu Leu Gln Lys Arg Gln Phe Phe His Ser His Arg Ala Gln Pro Met

485 490 495

Ala Leu Glu Gln Val Leu Ser Asp Arg Asp Ser Glu Asp Glu Val Asp

500 505 510

Asp Asp Val Ala Asp Phe Glu Asp Arg Arg Met Leu Asp Asp Phe Val

515 520 525

Asp Val Thr Lys Asp Glu Lys Gln Met Met His Met Trp Asn Ser Phe

530 535 540

Val Arg Lys Gln Arg Val Leu Ala Asp Gly His Ile Pro Trp Ala Cys

545 550 555 560

Glu Ala Phe Ser Arg Leu His Gly Pro Ile Met Val Arg Thr Pro His

565 570 575

Leu Ile Trp Cys Trp Arg Val Phe Met Val Lys Leu Trp Asn His Gly

580 585 590

Leu Leu Asp Ala Arg Thr Met Asn Asn Cys Asn Thr Phe Leu Glu Gln

595 600 605

Leu Gln Ile

610

<210> SEQ ID NO 2

<211> LENGTH: 2280

<212> TYPE: DNA

<213> ORGANISM: Arabidopsis thaliana

<220> FEATURE:

<221> NAME/KEY: CDS

<222> LOCATION: (310)..(2142)

<221> NAME/KEY: misc_feature

<222> LOCATION: (1650)..(1655)

<223> OTHER INFORMATION: BamHI recognition site

<221> NAME/KEY: misc_feature

<222> LOCATION: (1984)..(1989)

<223> OTHER INFORMATION: SphI recognition site

<400> SEQUENCE: 2

aagataattt ctcacaatta gggttttttt tttcttctga gttaactgtt ccatctccat 60

cctaatcttc accttctcct tgatttcgag atctctgtca atttgttgaa tctgttcttt 120

atctaattag ctcaactccg agtctttgct ggattttgaa gcttttgtag ctgaagcaaa 180

tttgtaatct gtgatggtgt atgcactgat tctgggtatg gtattgtact ctaggatctc 240

gtagcgagaa tgccaggcat tcctcttgtt agtcgtgaaa cctcttcttg ttcaagaagc 300

acagagcag atg tgc cat gaa gac tcc cgt ctg cgt att tcg gaa gag gag 351

Met Cys His Glu Asp Ser Arg Leu Arg Ile Ser Glu Glu Glu

1 5 10

gag att gct gct gaa gag agc ttg gct gcc tat tgc aag cct gtt gaa 399

Glu Ile Ala Ala Glu Glu Ser Leu Ala Ala Tyr Cys Lys Pro Val Glu

15 20 25 30

ctc tac aat atc att caa cgc cgt gct att agg aat ccc ttg ttt ctt 447

Leu Tyr Asn Ile Ile Gln Arg Arg Ala Ile Arg Asn Pro Leu Phe Leu

35 40 45

cag cga tgt ttg cat tat aag att gag gca aaa cat aaa agg aga ata 495

Gln Arg Cys Leu His Tyr Lys Ile Glu Ala Lys His Lys Arg Arg Ile

50 55 60

caa atg act gta ttc ctc tcg ggc gct ata gat gct ggg gta caa act 543

Gln Met Thr Val Phe Leu Ser Gly Ala Ile Asp Ala Gly Val Gln Thr

65 70 75

caa aaa tta ttc cct ctg tat att ttg ttg gca aga ctc gtt tct cct 591

Gln Lys Leu Phe Pro Leu Tyr Ile Leu Leu Ala Arg Leu Val Ser Pro

80 85 90

aag cct gtc gct gag tat tct gca gta tat agg ttc agt cga gca tgt 639

Lys Pro Val Ala Glu Tyr Ser Ala Val Tyr Arg Phe Ser Arg Ala Cys

95 100 105 110

atc cta act ggt gga ttg ggg gtt gat gga gtt agt caa gcc caa gcc 687

Ile Leu Thr Gly Gly Leu Gly Val Asp Gly Val Ser Gln Ala Gln Ala

115 120 125

aac ttt ctt ctc cct gat atg aat aga ctc gca ttg gag gca aaa tca 735

Asn Phe Leu Leu Pro Asp Met Asn Arg Leu Ala Leu Glu Ala Lys Ser

130 135 140

gga tca ctc gct atc ttg ttt atc agc ttt gct ggt gcg caa aat tct 783

Gly Ser Leu Ala Ile Leu Phe Ile Ser Phe Ala Gly Ala Gln Asn Ser

145 150 155

caa ttt ggc att gat tca ggc aag att cat tca gga aat ata gga gga 831

Gln Phe Gly Ile Asp Ser Gly Lys Ile His Ser Gly Asn Ile Gly Gly

160 165 170

cat tgt tta tgg agc aaa ata cct ctg caa tca ctg tat gcg tcg tgg 879

His Cys Leu Trp Ser Lys Ile Pro Leu Gln Ser Leu Tyr Ala Ser Trp

175 180 185 190

cag aaa tca cca aac atg gac ttg gga cag aga gta gac aca gtc tct 927

Gln Lys Ser Pro Asn Met Asp Leu Gly Gln Arg Val Asp Thr Val Ser

195 200 205

ctt gtt gaa atg cag cct tgc ttc ata aag cta aag tcc atg agt gag 975

Leu Val Glu Met Gln Pro Cys Phe Ile Lys Leu Lys Ser Met Ser Glu

210 215 220

gaa aag tgt gtc tcg att cag gtg ccc agc aat cca ctc acc tcg agc 1023

Glu Lys Cys Val Ser Ile Gln Val Pro Ser Asn Pro Leu Thr Ser Ser

225 230 235

tct ccg cag caa gtg caa gtc acc ata tct gca gaa gaa gtt ggg tca 1071

Ser Pro Gln Gln Val Gln Val Thr Ile Ser Ala Glu Glu Val Gly Ser

240 245 250

acg gaa aaa tct cct tat agt tca ttt tca tat aat gac atc tct tcc 1119

Thr Glu Lys Ser Pro Tyr Ser Ser Phe Ser Tyr Asn Asp Ile Ser Ser

255 260 265 270

tct tcc ttg ttg caa att atc agg ttg aga aca gga aat gta gtt ttc 1167

Ser Ser Leu Leu Gln Ile Ile Arg Leu Arg Thr Gly Asn Val Val Phe

275 280 285

aac tac aga tac tat aac aac aaa ttg cag aag act gaa gta act gaa 1215

Asn Tyr Arg Tyr Tyr Asn Asn Lys Leu Gln Lys Thr Glu Val Thr Glu

290 295 300

gac ttt tct tgt cca ttc tgc tta gta aaa tgt gcc agt ttc aag ggc 1263

Asp Phe Ser Cys Pro Phe Cys Leu Val Lys Cys Ala Ser Phe Lys Gly

305 310 315

ctg aga tat cac ttg cca tca acc cac gat ctc ctc aat ttc gag ttt 1311

Leu Arg Tyr His Leu Pro Ser Thr His Asp Leu Leu Asn Phe Glu Phe

320 325 330

tgg gta act gaa gaa ttt cag gcg gta aat gtc tcc ctc aag act gag 1359

Trp Val Thr Glu Glu Phe Gln Ala Val Asn Val Ser Leu Lys Thr Glu

335 340 345 350

aca atg ata tcc aag gtt aat gag gat gac gtt gac cca aag cag caa 1407

Thr Met Ile Ser Lys Val Asn Glu Asp Asp Val Asp Pro Lys Gln Gln

355 360 365

act ttc ttt ttt tct tcc aaa aaa ttc aga cgg agg agg caa aag agt 1455

Thr Phe Phe Phe Ser Ser Lys Lys Phe Arg Arg Arg Arg Gln Lys Ser

370 375 380

cag gta cgg agc tca agg caa ggg cct cat ctt gga tta ggt tgc gag 1503

Gln Val Arg Ser Ser Arg Gln Gly Pro His Leu Gly Leu Gly Cys Glu

385 390 395

gtg cta gat aag act gat gat gct cat tct gtt aga agt gag aag agc 1551

Val Leu Asp Lys Thr Asp Asp Ala His Ser Val Arg Ser Glu Lys Ser

400 405 410

cga ata cca cct gga aag cat tac gaa aga att ggg ggt gct gag tct 1599

Arg Ile Pro Pro Gly Lys His Tyr Glu Arg Ile Gly Gly Ala Glu Ser

415 420 425 430

ggt caa aga gtt cct cct ggc acg agt cct gca gac gtg caa tca tgt 1647

Gly Gln Arg Val Pro Pro Gly Thr Ser Pro Ala Asp Val Gln Ser Cys

435 440 445

ggg gat cca gat tat gtg cag tcg ata gct gga agt aca atg ttg cag 1695

Gly Asp Pro Asp Tyr Val Gln Ser Ile Ala Gly Ser Thr Met Leu Gln

450 455 460

ttt gca aaa acg agg aaa ata tct ata gaa cgg tcg gac ttg agg aac 1743

Phe Ala Lys Thr Arg Lys Ile Ser Ile Glu Arg Ser Asp Leu Arg Asn

465 470 475

cga agc ctc ctt cag aag aga cag ttc ttc cac tct cat cga gct cag 1791

Arg Ser Leu Leu Gln Lys Arg Gln Phe Phe His Ser His Arg Ala Gln

480 485 490

ccc atg gct cta gaa caa gta ctt tcg gac cgg gat agt gaa gat gaa 1839

Pro Met Ala Leu Glu Gln Val Leu Ser Asp Arg Asp Ser Glu Asp Glu

495 500 505 510

gtt gat gat gat gtg gca gat ttt gaa gat aga agg atg ctc gat gat 1887

Val Asp Asp Asp Val Ala Asp Phe Glu Asp Arg Arg Met Leu Asp Asp

515 520 525

ttc gtt gat gtg act aaa gat gag aaa cag atg atg cac atg tgg aac 1935

Phe Val Asp Val Thr Lys Asp Glu Lys Gln Met Met His Met Trp Asn

530 535 540

tcg ttt gtg agg aag cag cga gta tta gca gat ggt cac att cca tgg 1983

Ser Phe Val Arg Lys Gln Arg Val Leu Ala Asp Gly His Ile Pro Trp

545 550 555

gca tgc gag gca ttc tca aga ttg cac gga ccc atc atg gtt cga aca 2031

Ala Cys Glu Ala Phe Ser Arg Leu His Gly Pro Ile Met Val Arg Thr

560 565 570

ccg cac ttg att tgg tgc tgg aga gtg ttt atg gtg aaa ctg tgg aac 2079

Pro His Leu Ile Trp Cys Trp Arg Val Phe Met Val Lys Leu Trp Asn

575 580 585 590

cac ggt ctt ctt gat gcc cga acc atg aac aac tgt aat acc ttt ctc 2127

His Gly Leu Leu Asp Ala Arg Thr Met Asn Asn Cys Asn Thr Phe Leu

595 600 605

gaa cag ctc caa att tgaaaaccca agaaatcatt aatttaagta gaaaaacaaa 2182

Glu Gln Leu Gln Ile

610

gaaagacaag agaagaagag ttttgggttc tcatttaact acttttggtg ttttaagaga 2242

aagaggagca tatttatgca tgaaaaaaaa aaaaaaaa 2280

<210> SEQ ID NO 3

<211> LENGTH: 5580

<212> TYPE: DNA

<213> ORGANISM: Arabidopsis thaliana

<220> FEATURE:

<221> NAME/KEY: intron

<222> LOCATION: (162)..(267)

<221> NAME/KEY: intron

<222> LOCATION: (394)..(565)

<221> NAME/KEY: CDS

<222> LOCATION: (588)..(713)

<221> NAME/KEY: intron

<222> LOCATION: (714)..(930)

<221> NAME/KEY: CDS

<222> LOCATION: (931)..(986)

<221> NAME/KEY: intron

<222> LOCATION: (987)..(1132)

<221> NAME/KEY: CDS

<222> LOCATION: (1133)..(1247)

<221> NAME/KEY: intron

<222> LOCATION: (1248)..(1344)

<221> NAME/KEY: CDS

<222> LOCATION: (1345)..(1504)

<221> NAME/KEY: intron

<222> LOCATION: (1505)..(1596)

<221> NAME/KEY: CDS

<222> LOCATION: (1597)..(1647)

<221> NAME/KEY: intron

<222> LOCATION: (1648)..(1854)

<221> NAME/KEY: CDS

<222> LOCATION: (1855)..(1994)

<221> NAME/KEY: intron

<222> LOCATION: (1995)..(2197)

<221> NAME/KEY: CDS

<222> LOCATION: (2198)..(2260)

<221> NAME/KEY: intron

<222> LOCATION: (2261)..(2350)

<221> NAME/KEY: CDS

<222> LOCATION: (2351)..(2472)

<221> NAME/KEY: intron

<222> LOCATION: (2473)..(2714)

<221> NAME/KEY: CDS

<222> LOCATION: (2715)..(2779)

<221> NAME/KEY: intron

<222> LOCATION: (2780)..(2870)

<221> NAME/KEY: CDS

<222> LOCATION: (2871)..(2930)

<221> NAME/KEY: intron

<222> LOCATION: (2931)..(3038)

<221> NAME/KEY: CDS

<222> LOCATION: (3039)..(3092)

<221> NAME/KEY: intron

<222> LOCATION: (3093)..(3174)

<221> NAME/KEY: CDS

<222> LOCATION: (3175)..(3234)

<221> NAME/KEY: intron

<222> LOCATION: (3235)..(3654)

<221> NAME/KEY: CDS

<222> LOCATION: (3656)..(3701)

<221> NAME/KEY: intron

<222> LOCATION: (3702)..(3784)

<221> NAME/KEY: CDS

<222> LOCATION: (3785)..(3885)

<221> NAME/KEY: intron

<222> LOCATION: (3886)..(4052)

<221> NAME/KEY: CDS

<222> LOCATION: (4053)..(4272)

<221> NAME/KEY: intron

<222> LOCATION: (4273)..(4428)

<221> NAME/KEY: CDS

<222> LOCATION: (4429)..(4477)

<221> NAME/KEY: intron

<222> LOCATION: (4478)..(4552)

<221> NAME/KEY: CDS

<222> LOCATION: (4553)..(4636)

<221> NAME/KEY: intron

<222> LOCATION: (4637)..(4982)

<221> NAME/KEY: CDS

<222> LOCATION: (4983)..(5062)

<221> NAME/KEY: intron

<222> LOCATION: (5063)..(5265)

<221> NAME/KEY: CDS

<222> LOCATION: (5266)..(5355)

<221> NAME/KEY: intron

<222> LOCATION: (5356)..(5445)

<221> NAME/KEY: CDS

<222> LOCATION: (5446)..(5542)

<400> SEQUENCE: 3

aagataattt ctcacaatta gggttttttt tttcttctga gttaactgtt ccatctccat 60

cctaatcttc accttctcct tgatttcgag atctctgtca atttgttgaa tctgttcttt 120

atctaattag ctcaactccg agtctttgct ggattttgaa ggtcaccact gttcaagttt 180

acattttttt tcctgctaat cgcttgatac ccgtttctgc tgttgtggga tttattgggt 240

ttttcttctt tacgattttt gttgcagctt ttgtagctga agcaaatttg taatctgtga 300

tggtgtatgc actgattctg ggtatggtat tgtactctag gatctcgtag cgagaatgcc 360

aggcattcct cttgttagtc gtgaaacctc ttcgtaagtc tcatgaacaa cctaatgctt 420

ctataatgtc tctgcagcat tgtgtaactt tatactgttt ctcttatgta taagctgagg 480

aatcctagta attcaaactt atcaaatttt tattttgttg tggggttgct tacaattttg 540

gttgcgtatg atggtgaaat cacagttgtt caagaagcac agagcag atg tgc cat 596

Met Cys His

1

gaa gac tcc cgt ctg cgt att tcg gaa gag gag gag att gct gct gaa 644

Glu Asp Ser Arg Leu Arg Ile Ser Glu Glu Glu Glu Ile Ala Ala Glu

5 10 15

gag agc ttg gct gcc tat tgc aag cct gtt gaa ctc tac aat atc att 692

Glu Ser Leu Ala Ala Tyr Cys Lys Pro Val Glu Leu Tyr Asn Ile Ile

20 25 30 35

caa cgc cgt gct att agg aat gtatgtcttc cttcctacct tttttagaca gaat 747

Gln Arg Arg Ala Ile Arg Asn

40

atgtttagtt atgacttatg agctcagctg atatatcaca tgtattggtt tacttttgag 807

ttttgacaat gaaaatttac atgaaaatgt agtttgagtt gacttcattt ggtataagca 867

agtatgtgtt gtcttgctat gcagtccatc ctaatcattt ctctctctct gtctcccctg 927

tag ccc ttg ttt ctt cag cga tgt ttg cat tat aag att gag gca aaa 975

Pro Leu Phe Leu Gln Arg Cys Leu His Tyr Lys Ile Glu Ala Lys

45 50 55

cat aaa agg ag gtaagctttt tttttttcct tcctttctct gttcagaatc tccatt 1032

His Lys Arg Arg

60

acttttgggt aactattaca ctatacctta gtaattcatt ccggacttga atgctttcta 1092

agttttcgga tagttatcaa tatatattac tgctttgcag a ata caa atg act gta 1148

Ile Gln Met Thr Val

65

ttc ctc tcg ggc gct ata gat gct ggg gta caa act caa aaa tta ttc 1196

Phe Leu Ser Gly Ala Ile Asp Ala Gly Val Gln Thr Gln Lys Leu Phe

70 75 80

cct ctg tat att ttg ttg gca aga ctc gtt tct cct aag cct gtc gct 1244

Pro Leu Tyr Ile Leu Leu Ala Arg Leu Val Ser Pro Lys Pro Val Ala

85 90 95

gag gtatgcattt gaacctcaga cagatttgca ttgatcttta ttatttgtaa cttacc 1303

Glu

tattctttgc taacattttt cttgaaattc tcaaattata g tat tct gca gta tat 1359

Tyr Ser Ala Val Tyr

100

agg ttc agt cga gca tgt atc cta act ggt gga ttg ggg gtt gat gga 1407

Arg Phe Ser Arg Ala Cys Ile Leu Thr Gly Gly Leu Gly Val Asp Gly

105 110 115 120

gtt agt caa gcc caa gcc aac ttt ctt ctc cct gat atg aat aga ctc 1455

Val Ser Gln Ala Gln Ala Asn Phe Leu Leu Pro Asp Met Asn Arg Leu

125 130 135

gca ttg gag gca aaa tca gga tca ctc gct atc ttg ttt atc agc ttt g 1504

Ala Leu Glu Ala Lys Ser Gly Ser Leu Ala Ile Leu Phe Ile Ser Phe

140 145 150

gtgattaaga ctgactgtgt acaaaattat ataaagacat ttatatatgt acagtattca 1564

gataaactga tcacataatt ttcttcttgt ag ct ggt gcg caa aat tct caa 1616

Ala Gly Ala Gln Asn Ser Gln

155

ttt ggc att gat tca ggc aag att cat tca g gtacttccat ttcttcattg a 1668

Phe Gly Ile Asp Ser Gly Lys Ile His Ser

160 165

tataacattc taatattgaa aagttatgta tctttgggca ttaccaattt tccatgtaat 1728

agtatggaaa atctcagtcc tatttattaa caaaagaatt agggattctt tgactccaat 1788

tataagagtt tctgaaagtc ttttttttca ttaactctta ccatcggaag cgtttttttc 1848

tgccag ga aat ata gga gga cat tgt tta tgg agc aaa ata cct ctg caa 1898

Gly Asn Ile Gly Gly His Cys Leu Trp Ser Lys Ile Pro Leu Gln

170 175 180

tca ctg tat gcg tcg tgg cag aaa tca cca aac atg gac ttg gga cag 1946

Ser Leu Tyr Ala Ser Trp Gln Lys Ser Pro Asn Met Asp Leu Gly Gln

185 190 195 200

aga gta gac aca gtc tct ctt gtt gaa atg cag cct tgc ttc ata aag g 1995

Arg Val Asp Thr Val Ser Leu Val Glu Met Gln Pro Cys Phe Ile Lys

205 210 215

taaacactat tgcccaagtc ttcctcttgt tctatgactt tatgctccct gtattgaaat 2055

aaggactgtg tattgaactt cttttgttat ttgaaaaagt aaattggaag taattgctac 2115

tgtgaatttt atttttgcca ttagttttca gtcttgatta tttaaatgaa aatattacgg 2175

tataacttgt ccattgctgc ag cta aag tcc atg agt gag gaa aag tgt gtc 2227

Leu Lys Ser Met Ser Glu Glu Lys Cys Val

220 225

tcg att cag gtg ccc agc aat cca ctc acc tcg gtaactttgc acactttgct 2280

Ser Ile Gln Val Pro Ser Asn Pro Leu Thr Ser

230 235

atacttccat acattattct gaaatatcat gtaatcatat tcttacaatt cttacacttc 2340

ttatttgaag agc tct ccg cag caa gtg caa gtc acc ata tct gca gaa 2389

Ser Ser Pro Gln Gln Val Gln Val Thr Ile Ser Ala Glu

240 245 250

gaa gtt ggg tca acg gaa aaa tct cct tat agt tca ttt tca tat aat 2437

Glu Val Gly Ser Thr Glu Lys Ser Pro Tyr Ser Ser Phe Ser Tyr Asn

255 260 265

gac atc tct tcc tct tcc ttg ttg caa att atc ag gtaatcttca gtttagt 2489

Asp Ile Ser Ser Ser Ser Leu Leu Gln Ile Ile Arg

270 275

ctgcaatttc ttctgcgctc tcagatttct tgcctcatct cattatgatt ttttgtaatt 2549

gtataaaata tattggccgg tctgctatct cccttaatat atagttggca gttttcttga 2609

attgtgactg tcctcctctt ttatggggat tatacaagtc gttacgtaca actaaaaatg 2669

tccatctcgt taagttgact ctataccact acattcattg catag g ttg aga aca 2724

Leu Arg Thr

280

gga aat gta gtt ttc aac tac aga tac tat aac aac aaa ttg cag aag 2772

Gly Asn Val Val Phe Asn Tyr Arg Tyr Tyr Asn Asn Lys Leu Gln Lys

285 290 295

act gaa g gtaactagta ttattttaac ctgtttcata cccatgtgtg tctatatttc 2829

Thr Glu

atccgttacc ctaacctgtt acgtatatgt ttgctatgtg tcttgcag ta act gaa 2885

Val Thr Glu

300

gac ttt tct tgt cca ttc tgc tta gta aaa tgt gcc agt ttc aag gtgga 2935

Asp Phe Ser Cys Pro Phe Cys Leu Val Lys Cys Ala Ser Phe Lys

305 310 315

ctttcatttc cattctcatt catcctctta gtcaaagata cagctgtagt gactagtctt 2995

tgtagtgatg caatcttttc tttttctccc aatcatgttg tag ggc ctg aga tat 3050

Gly Leu Arg Tyr

320

cac ttg cca tca acc cac gat ctc ctc aat ttc gag ttt tgg gttgtagct 3101

His Leu Pro Ser Thr His Asp Leu Leu Asn Phe Glu Phe Trp

325 330 335

ttaaaattca gttaacctgt ttgatctttt ttttttattt tgtgggtgcc actaatctgc 3161

tttacttggt tag gta act gaa gaa ttt cag gcg gta aat gtc tcc ctc 3210

Val Thr Glu Glu Phe Gln Ala Val Asn Val Ser Leu

340 345

aag act gag aca atg ata tcc aag gttagaacat cttgtttgtt cgatttatgt 3264

Lys Thr Glu Thr Met Ile Ser Lys

350 355

tcattagttt ctctgctgta tatcttatag gctgtaacaa attcattttt catttaaact 3324

aatatcctcc atgggttgtt gacttttgtg tggttaaata agggaactgg aatctttagt 3384

tgctatttgt cacactatga tccttgctat tgtccttaat agcgtgatga gaataaactc 3444

aaaatgacat cgctgttctg tttacttttt gtggccatga gaccgtcaaa gctcgactgt 3504

agaataaagt cctggattat ataggagtgt caaatctaat tgaagtagtt ggttctacaa 3564

tatattctat gtctttgtag tttttcctat ttgatgatta ctcttagcac agttttctaa 3624

atgttaatgt tcattaaaaa atctgctcag gtt aat gag gat gac gtt gac cca 3678

Val Asn Glu Asp Asp Val Asp Pro

360

aag cag caa act ttc ttt ttt tc gtaagttatc tggcctatat gttgcctttt 3731

Lys Gln Gln Thr Phe Phe Phe Ser

365 370

attatctttc cagcatctgt gtgagaccat aaaaattctt caatatgtga cag t tcc 3788

Ser

aaa aaa ttc aga cgg agg agg caa aag agt cag gta cgg agc tca agg 3836

Lys Lys Phe Arg Arg Arg Arg Gln Lys Ser Gln Val Arg Ser Ser Arg

375 380 385

caa ggg cct cat ctt gga tta ggt tgc gag gtg cta gat aag act gat g 3885

Gln Gly Pro His Leu Gly Leu Gly Cys Glu Val Leu Asp Lys Thr Asp

390 395 400

gtatgtgttt gactgaaatg acagttaatt ggatttgtag tattggcttc ttttgtgatg 3945

agagcctgtc ttagttgtat attttacgag tattttactt tgttatgtgc aattttgcat 4005

gcaacaacgt tggatcattt ggcacagctt tttattctta ctttcag at gct cat 4060

Asp Ala His

405

tct gtt aga agt gag aag agc cga ata cca cct gga aag cat tac gaa 4108

Ser Val Arg Ser Glu Lys Ser Arg Ile Pro Pro Gly Lys His Tyr Glu

410 415 420

aga att ggg ggt gct gag tct ggt caa aga gtt cct cct ggc acg agt 4156

Arg Ile Gly Gly Ala Glu Ser Gly Gln Arg Val Pro Pro Gly Thr Ser

425 430 435

cct gca gac gtg caa tca tgt ggg gat cca gat tat gtg cag tcg ata 4204

Pro Ala Asp Val Gln Ser Cys Gly Asp Pro Asp Tyr Val Gln Ser Ile

440 445 450 455

gct gga agt aca atg ttg cag ttt gca aaa acg agg aaa ata tct ata 4252

Ala Gly Ser Thr Met Leu Gln Phe Ala Lys Thr Arg Lys Ile Ser Ile

460 465 470

gaa cgg tcg gac ttg agg aa gtatgtttga cttccttttg tcgttctatc ctctt 4307

Glu Arg Ser Asp Leu Arg Asn

475

cttcaattta tatttaacta catatggttc atgcatgaaa aattgtgtcc tagttttata 4367

acaagtagct tgttaatccc aaatgatgtg agtgagtttt tcaaattttt tcctcctcca 4427

g c cga agc ctc ctt cag aag aga cag ttc ttc cac tct cat cga gct 4474

Arg Ser Leu Leu Gln Lys Arg Gln Phe Phe His Ser His Arg Ala

480 485 490

cag gtgatctttt ttctttagct ctcttgcttt tgaagattgc aattgatttt gacttt 4533

Gln

gctatgtgta ctatcgcag ccc atg gct cta gaa caa gta ctt tcg gac cgg 4585

Pro Met Ala Leu Glu Gln Val Leu Ser Asp Arg

495 500 505

gat agt gaa gat gaa gtt gat gat gat gtg gca gat ttt gaa gat aga 4633

Asp Ser Glu Asp Glu Val Asp Asp Asp Val Ala Asp Phe Glu Asp Arg

510 515 520

agg gtatgttttt gaatttaata ttttcaccgc atcagtagtt gggtagaata aagctc 4692

Arg

agtagttggg tagatatatg tttcatgtga aagggaaagg aatattgaag actgggcatg 4752

ggcaaacgtt aggagcaata ttgtaatggt tcagagatca atagaaaata tgtgagcaag 4812

cctcacggtt tgatatggaa cagtagaacc agatcattag tgcttatata acactcatta 4872

aaagacgaag tgtgtccgtt tgtactcgat tctaacatag ttgattctaa catagtttgt 4932

ctgattctcc atatagtgaa taacgttatt tcctattact attctttcag atg ctc 4988

Met Leu

gat gat ttc gtt gat gtg act aaa gat gag aaa cag atg atg cac atg 5036

Asp Asp Phe Val Asp Val Thr Lys Asp Glu Lys Gln Met Met His Met

525 530 535 540

tgg aac tcg ttt gtg agg aag cag cg gtatgtctta tctcttttca gtacatgt 5090

Trp Asn Ser Phe Val Arg Lys Gln Arg

545

cacgtggagt tttccagtat aaacatttag agtcgcgcat gtaaaggttg tggataattc 5150

ctgcctgggt tcttctggtt aaaaaaaaaa aactgaacaa ttagataaca tacgcatcca 5210

tgttctctga ctcattataa gcattacctt gacagtggtt ttggaccctt tgcag a 5266

gta tta gca gat ggt cac att cca tgg gca tgc gag gca ttc tca aga 5314

Val Leu Ala Asp Gly His Ile Pro Trp Ala Cys Glu Ala Phe Ser Arg

550 555 560 565

ttg cac gga ccc atc atg gtt cga aca ccg cac ttg att tg gtaattcaac 5365

Leu His Gly Pro Ile Met Val Arg Thr Pro His Leu Ile Trp

570 575

tctcatttct tccattgttt tttccagtgt atcggagaag aaagcggttt tgttgataaa 5425

agtgagcttt ttttgtgtag g tgc tgg aga gtg ttt atg gtg aaa ctg tgg 5476

Cys Trp Arg Val Phe Met Val Lys Leu Trp

580 585

aac cac ggt ctt ctt gat gcc cga acc atg aac aac tgt aat acc ttt 5524

Asn His Gly Leu Leu Asp Ala Arg Thr Met Asn Asn Cys Asn Thr Phe

590 595 600 605

ctc gaa cag ctc caa att tgaaaaccca agaaatcatt aatttaagta gaaaaaca 5580

Leu Glu Gln Leu Gln Ile

610

<210> SEQ ID NO 4

<211> LENGTH: 23

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial Sequence:Artificially

Synthesized Oligonucleotide Primer Sequence

<400> SEQUENCE: 4

ggatccgaac ccgactcggt acc 23

<210> SEQ ID NO 5

<211> LENGTH: 24

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial Sequence:Artificially

Synthesized Oligonucleotide Primer Sequence

<400> SEQUENCE: 5

gcttatggat gtggactctc taac 24

<210> SEQ ID NO 6

<211> LENGTH: 22

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial Sequence:Artificially

Synthesized Oligonucleotide Primer Sequence

<400> SEQUENCE: 6

aggtcctaca actacaacag tt 22

<210> SEQ ID NO 7

<211> LENGTH: 22

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial Sequence:Artificially

Synthesized Oligonucleotide Primer Sequence

<400> SEQUENCE: 7

gaggaagcta gtattctctt tg 22

<210> SEQ ID NO 8

<211> LENGTH: 604

<212> TYPE: PRT

<213> ORGANISM: Oryza sativa

<220> FEATURE:

<221> NAME/KEY: ZN_FING

<222> LOCATION: (310)..(335)

<400> SEQUENCE: 8

Met Cys Arg His Gln Pro Arg Ala Arg Leu Ser Pro Asp Glu Gln Leu

1 5 10 15

Ala Ala Glu Glu Ser Phe Ala Leu Tyr Cys Lys Pro Val Glu Leu Tyr

20 25 30

Asn Ile Ile Gln Arg Arg Ser Ile Lys Asn Pro Ala Phe Leu Gln Arg

35 40 45

Cys Leu Leu Tyr Lys Ile His Ala Arg Arg Lys Lys Arg Ser Leu Ile

50 55 60

Thr Ile Ser Leu Ser Gly Gly Thr Asn Lys Glu Leu Arg Ala Gln Asn

65 70 75 80

Ile Phe Pro Leu Tyr Val Leu Leu Ala Arg Pro Thr Asn Asn Val Ser

85 90 95

Leu Glu Gly His Ser Pro Ile Tyr Arg Phe Ser Arg Ala Cys Leu Leu

100 105 110

Thr Ser Phe His Glu Phe Gly Asn Lys Asp Tyr Thr Glu Ala Thr Phe

115 120 125

Val Ile Pro Asp Val Lys Asn Leu Ala Thr Ser Arg Ala Cys Ser Leu

130 135 140

Asn Ile Ile Leu Ile Ser Cys Gly Arg Ala Glu Gln Thr Phe Asp Asp

145 150 155 160

Asn Asn Cys Ser Gly Asn His Val Glu Gly Ser Thr Leu Gln Lys Leu

165 170 175

Glu Gly Lys Cys Phe Trp Gly Lys Ile Pro Ile Asp Leu Leu Ala Ser

180 185 190

Ser Leu Gly Asn Cys Val Ser Leu Ser Leu Gly His Thr Val Glu Met

195 200 205

Ser Ser Thr Val Glu Met Thr Pro Ser Phe Leu Glu Pro Lys Phe Leu

210 215 220

Glu Asp Asp Ser Cys Leu Thr Phe Cys Ser Gln Lys Val Asp Ala Thr

225 230 235 240

Gly Ser Phe Gln Leu Gln Val Ser Ile Ser Ala Gln Glu Ala Gly Ala

245 250 255

Lys Asp Met Ser Glu Ser Pro Tyr Ser Val Tyr Ser Tyr Asn Asp Val

260 265 270

Pro Pro Ser Ser Leu Thr His Ile Ile Arg Leu Arg Ser Gly Asn Val

275 280 285

Leu Phe Asn Tyr Lys Tyr Tyr Asn Asn Thr Met Gln Lys Thr Glu Val

290 295 300

Thr Glu Asp Phe Ser Cys Pro Phe Cys Leu Val Pro Cys Gly Ser Phe

305 310 315 320

Lys Gly Leu Gly Cys His Leu Asn Ala Ser His Asp Leu Phe His Tyr

325 330 335

Glu Phe Trp Ile Ser Glu Glu Cys Gln Ala Val Asn Val Ser Leu Lys

340 345 350

Thr Asp Ser Trp Arg Thr Glu Leu Leu Ala Glu Gly Val Asp Pro Arg

355 360 365

His Gln Thr Phe Ser Tyr Arg Ser Arg Phe Lys Lys Arg Lys Arg Val

370 375 380

Glu Ile Ser Ser Asp Lys Ile Arg His Val His Pro His Ile Val Asp

385 390 395 400

Ser Gly Ser Pro Glu Asp Ala Gln Ala Gly Ser Glu Asp Asp Tyr Val

405 410 415

Gln Arg Glu Asn Gly Ser Ser Val Ala His Ala Ser Val Asp Pro Ala

420 425 430

Asn Ser Leu His Gly Ser Asn Leu Ser Ala Pro Thr Val Leu Gln Phe

435 440 445

Gly Lys Thr Arg Lys Leu Ser Val Glu Arg Ala Asp Pro Arg Asn Arg

450 455 460

Gln Leu Leu Gln Lys Arg Gln Phe Phe His Ser His Arg Ala Gln Pro

465 470 475 480

Met Ala Trp Ser Lys Val Phe Ser Asp Arg Asp Ser Glu Asp Glu Val

485 490 495

Asp Asp Asp Ile Ala Asp Phe Glu Asp Arg Arg Met Leu Asp Asp Phe

500 505 510

Val Asp Val Thr Lys Asp Glu Lys Leu Ile Met His Met Trp Asn Ser

515 520 525

Phe Val Arg Lys Gln Arg Val Leu Ala Asp Gly His Ile Pro Trp Ala

530 535 540

Cys Glu Ala Phe Ser Gln Phe His Gly Gln Glu Leu Val Gln Asn Pro

545 550 555 560

Ala Leu Leu Trp Cys Trp Arg Phe Phe Met Val Lys Leu Trp Asn His

565 570 575

Ser Leu Leu Asp Ala Arg Ala Met Asn Ala Cys Asn Thr Ile Leu Glu

580 585 590

Gly Tyr Leu Asn Gly Ser Ser Asp Pro Lys Lys Asn

595 600

<210> SEQ ID NO 9

<211> LENGTH: 2248

<212> TYPE: DNA

<213> ORGANISM: Oryza sativa

<220> FEATURE:

<221> NAME/KEY: CDS

<222> LOCATION: (86)..(1897)

<221> NAME/KEY: misc_feature

<222> LOCATION: (36)..(41)

<223> OTHER INFORMATION: NheI recognition site

<400> SEQUENCE: 9

cgccgatccc catccctccc gcgagcagga gcagggctag ccgtcgttcc tcctgctgct 60

tccgccgcat ccatcctgat accag atg tgc cgc cac cag cca agg gct cgg 112

Met Cys Arg His Gln Pro Arg Ala Arg

1 5

ctc tct ccc gat gag cag ctt gca gct gaa gaa agc ttc gca tta tac 160

Leu Ser Pro Asp Glu Gln Leu Ala Ala Glu Glu Ser Phe Ala Leu Tyr

10 15 20 25

tgc aag ccg gtc gag ttg tat aat atc att cag cgc cga tcc att aaa 208

Cys Lys Pro Val Glu Leu Tyr Asn Ile Ile Gln Arg Arg Ser Ile Lys

30 35 40

aat cct gct ttt ctt caa aga tgc ctt ctt tac aag att cac gca aga 256

Asn Pro Ala Phe Leu Gln Arg Cys Leu Leu Tyr Lys Ile His Ala Arg

45 50 55

cgg aag aag agg agc ctg ata acc ata tca ctt tct gga ggc aca aat 304

Arg Lys Lys Arg Ser Leu Ile Thr Ile Ser Leu Ser Gly Gly Thr Asn

60 65 70

aaa gaa ctg cgg gca caa aat atc ttt cct ctt tat gtt ctg tta gct 352

Lys Glu Leu Arg Ala Gln Asn Ile Phe Pro Leu Tyr Val Leu Leu Ala

75 80 85

aga cct act aat aat gtt tca ctt gaa ggg cat tct ccg ata tat cga 400

Arg Pro Thr Asn Asn Val Ser Leu Glu Gly His Ser Pro Ile Tyr Arg

90 95 100 105

ttc agt cgt gct tgt ttg ttg act tct ttt cat gaa ttt gga aat aaa 448

Phe Ser Arg Ala Cys Leu Leu Thr Ser Phe His Glu Phe Gly Asn Lys

110 115 120

gac tac act gaa gca aca ttc gtc att cct gat gtg aag aac tta gca 496

Asp Tyr Thr Glu Ala Thr Phe Val Ile Pro Asp Val Lys Asn Leu Ala

125 130 135

acc tcc cga gct tgc agc ctt aat att atc ctt atc agc tgt gga cga 544

Thr Ser Arg Ala Cys Ser Leu Asn Ile Ile Leu Ile Ser Cys Gly Arg

140 145 150

gct gag caa act ttt gat gac aat aac tgt tct ggg aac cat gtg gaa 592

Ala Glu Gln Thr Phe Asp Asp Asn Asn Cys Ser Gly Asn His Val Glu

155 160 165

ggc tct act ctc caa aag ctt gaa ggg aag tgt ttc tgg ggt aaa ata 640

Gly Ser Thr Leu Gln Lys Leu Glu Gly Lys Cys Phe Trp Gly Lys Ile

170 175 180 185

cca atc gat ctt ctt gct tca tct ttg gga aat tgt gtg agc tta agt 688

Pro Ile Asp Leu Leu Ala Ser Ser Leu Gly Asn Cys Val Ser Leu Ser

190 195 200

ttg gga cat acc gtg gaa atg tct tcc acg gtt gag atg acc cca agc 736

Leu Gly His Thr Val Glu Met Ser Ser Thr Val Glu Met Thr Pro Ser

205 210 215

ttc tta gag cca aaa ttt ctg gag gat gac agt tgc ttg aca ttt tgc 784

Phe Leu Glu Pro Lys Phe Leu Glu Asp Asp Ser Cys Leu Thr Phe Cys

220 225 230

tct cag aag gtt gat gct act ggt tca ttt caa ctg caa gtt agc ata 832

Ser Gln Lys Val Asp Ala Thr Gly Ser Phe Gln Leu Gln Val Ser Ile

235 240 245

tct gct caa gag gct ggt gca aaa gac atg tcc gag tct cct tat agt 880

Ser Ala Gln Glu Ala Gly Ala Lys Asp Met Ser Glu Ser Pro Tyr Ser

250 255 260 265

gtt tat tca tat aat gat gtg cca cct tcg tca ttg aca cat att ata 928

Val Tyr Ser Tyr Asn Asp Val Pro Pro Ser Ser Leu Thr His Ile Ile

270 275 280

agg ttg aga tct ggc aat gtg ctt ttt aac tac aaa tac tac aat aat 976

Arg Leu Arg Ser Gly Asn Val Leu Phe Asn Tyr Lys Tyr Tyr Asn Asn

285 290 295

act atg caa aaa acc gaa gtc act gaa gat ttt tct tgc cca ttt tgc 1024

Thr Met Gln Lys Thr Glu Val Thr Glu Asp Phe Ser Cys Pro Phe Cys

300 305 310

ttg gta cca tgt ggc agc ttt aag ggt cta gga tgt cac cta aac gca 1072

Leu Val Pro Cys Gly Ser Phe Lys Gly Leu Gly Cys His Leu Asn Ala

315 320 325

tcg cat gac ctt ttc cat tat gag ttt tgg ata tct gaa gag tgc cag 1120

Ser His Asp Leu Phe His Tyr Glu Phe Trp Ile Ser Glu Glu Cys Gln

330 335 340 345

gct gtt aat gtt agt ctg aag act gat tct tgg aga aca gag ctt ttg 1168

Ala Val Asn Val Ser Leu Lys Thr Asp Ser Trp Arg Thr Glu Leu Leu

350 355 360

gct gag gga gtt gat cca aga cat caa aca ttt tcg tac cgc tca aga 1216

Ala Glu Gly Val Asp Pro Arg His Gln Thr Phe Ser Tyr Arg Ser Arg

365 370 375

ttt aag aag cgt aaa agg gtg gaa atc tca agt gat aaa att agg cat 1264

Phe Lys Lys Arg Lys Arg Val Glu Ile Ser Ser Asp Lys Ile Arg His

380 385 390

gta cat cca cat att gtg gat tca gga tca cct gaa gat gcc cag gca 1312

Val His Pro His Ile Val Asp Ser Gly Ser Pro Glu Asp Ala Gln Ala

395 400 405

gga tct gaa gac gat tac gtg cag agg gaa aat ggt agt tct gta gca 1360

Gly Ser Glu Asp Asp Tyr Val Gln Arg Glu Asn Gly Ser Ser Val Ala

410 415 420 425

cac gct tct gtt gat cct gct aat tca tta cac ggt agc aat ctt tca 1408

His Ala Ser Val Asp Pro Ala Asn Ser Leu His Gly Ser Asn Leu Ser

430 435 440

gca cca aca gtg tta cag ttt ggg aag aca aga aag ctg tct gtt gaa 1456

Ala Pro Thr Val Leu Gln Phe Gly Lys Thr Arg Lys Leu Ser Val Glu

445 450 455

cga gct gat ccc aga aat cgg cag ctc cta caa aaa cgc cag ttc ttt 1504

Arg Ala Asp Pro Arg Asn Arg Gln Leu Leu Gln Lys Arg Gln Phe Phe

460 465 470

cat tct cac agg gct caa cca atg gca tgg agc aaa gtt ttc tca gat 1552

His Ser His Arg Ala Gln Pro Met Ala Trp Ser Lys Val Phe Ser Asp

475 480 485

cgt gat agt gaa gat gaa gtt gat gat gac att gct gat ttt gaa gat 1600

Arg Asp Ser Glu Asp Glu Val Asp Asp Asp Ile Ala Asp Phe Glu Asp

490 495 500 505

aga aga atg ctt gat gat ttt gtt gat gtt aca aaa gac gag aaa ctt 1648

Arg Arg Met Leu Asp Asp Phe Val Asp Val Thr Lys Asp Glu Lys Leu

510 515 520

att atg cat atg tgg aat tca ttt gtt cgg aaa caa agg gta cta gcg 1696

Ile Met His Met Trp Asn Ser Phe Val Arg Lys Gln Arg Val Leu Ala

525 530 535

gat ggc cat att ccc tgg gca tgc gaa gca ttc tcg cag ttt cat gga 1744

Asp Gly His Ile Pro Trp Ala Cys Glu Ala Phe Ser Gln Phe His Gly

540 545 550

caa gaa ctt gta caa aat cca gct cta cta tgg tgt tgg agg ttt ttt 1792

Gln Glu Leu Val Gln Asn Pro Ala Leu Leu Trp Cys Trp Arg Phe Phe

555 560 565

atg gtc aaa ctc tgg aac cac agt cta ctg gat gcg cga gcc atg aat 1840

Met Val Lys Leu Trp Asn His Ser Leu Leu Asp Ala Arg Ala Met Asn

570 575 580 585

gcc tgc aac aca att ctt gaa ggc tac ctg aac gga agc tcg gat cca 1888

Ala Cys Asn Thr Ile Leu Glu Gly Tyr Leu Asn Gly Ser Ser Asp Pro

590 595 600

aag aaa aat tgacgcatac aaatcattgg ccaacctgta gagtaaaatg 1937

Lys Lys Asn

cacttgtact ggttctggcc attccaatag tttgttttgt ttttggaaaa aaagatgtct 1997

gaagaattga aagctaacat gtgttttgga gggaagaaaa ttgaaggctg gggcggtcat 2057

tgtttcattt agaactcttc tcgattctat ttattgtaat tgatgttact cataactgta 2117

gagcagtatc aagaccaaac tgtaatgata tggttagcaa tatttacata aaagtttatt 2177

ttgtttgttg tttagcaccg tgggcagaca atttaattcc tatgcaggcc ctttttcatc 2237

gtcaaaaaaa a 2248

<210> SEQ ID NO 10

<211> LENGTH: 24

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial Sequence:Artificially

Synthesized Oligonucleotide Primer Sequence

<400> SEQUENCE: 10

gacgagaaac ttattatgca tatg 24

<210> SEQ ID NO 11

<211> LENGTH: 26

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial Sequence:Artificially

Synthesized Oligonucleotide Primer Sequence

<400> SEQUENCE: 11

ggtcttgata ctgctctaca gttatg 26

<210> SEQ ID NO 12

<211> LENGTH: 7

<212> TYPE: PRT

<213> ORGANISM: Arabidopsis thaliana

<400> SEQUENCE: 12

Lys Arg Gln Phe Phe His Ser

1 5

<210> SEQ ID NO 13

<211> LENGTH: 6

<212> TYPE: PRT

<213> ORGANISM: Arabidopsis thaliana

<400> SEQUENCE: 13

Trp Ala Cys Glu Ala Phe

1 5

<210> SEQ ID NO 14

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial Sequence:Artificially

Synthesized Oligonucleotide Primer Sequence

<400> SEQUENCE: 14

aagcggcaat tttaycaytc 20

<210> SEQ ID NO 15

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial Sequence:Artificially

Synthesized Oligonucleotide Primer Sequence

<400> SEQUENCE: 15

aagcggcagt tctaycaytc 20

<210> SEQ ID NO 16

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial Sequence:Artificially

Synthesized Oligonucleotide Primer Sequence

<400> SEQUENCE: 16

aagcggcagt tctaycayag 20

<210> SEQ ID NO 17

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial Sequence:Artificially

Synthesized Oligonucleotide Primer Sequence

<400> SEQUENCE: 17

aagcggcaat tttaycayag 20

<210> SEQ ID NO 18

<211> LENGTH: 17

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial Sequence:Artificially

Synthesized Oligonucleotide Primer Sequence

<221> NAME/KEY: misc-feature

<222> LOCATION: 12

<223> OTHER INFORMATION: n is a or t or c or g

<400> SEQUENCE: 18

aatacctcac angccca 17

<210> SEQ ID NO 19

<211> LENGTH: 17

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Description of Artificial Sequence:Artificially

Synthesized Oligonucleotide Primer Sequence

<221> NAME/KEY: misc-feature

<222> LOCATION: 12

<223> OTHER INFORMATION: n is a or t or c or g

<400> SEQUENCE: 19

aatacttcgc angccca 17

<210> SEQ ID NO 20

<211> LENGTH: 1216

<212> TYPE: DNA

<213> ORGANISM: Beta vulgaris

<220> FEATURE:

<221> NAME/KEY: misc_feature

<222> LOCATION: (1)..(20)

<223> OTHER INFORMATION: Oligonucleotide Primer “KR1” Sequence

<221> NAME/KEY: CDS

<222> LOCATION: (21)..(33)

<221> NAME/KEY: intron

<222> LOCATION: (34)..(694)

<221> NAME/KEY: CDS

<222> LOCATION: (695)..(778)

<221> NAME/KEY: intron

<222> LOCATION: (779)..(951)

<221> NAME/KEY: CDS

<222> LOCATION: (952)..(1031)

<221> NAME/KEY: intron

<222> LOCATION: (1032)..(1174)

<221> NAME/KEY: CDS

<222> LOCATION: (1175)..(1199)

<221> NAME/KEY: misc_feature

<222> LOCATION: (1200)..(1216)

<223> OTHER INFORMATION: Oligonucleotide Primer “WA2” Sequence

<400> SEQUENCE: 20

aagcggcaat tttatcattc t cac aga gct cag gtaatcaact gcagaagtca tat 56

His Arg Ala Gln

cgtgttatgc tgatgtctga actcctataa tataacagtt gttgactctt tgtttcctat 116

agtagttgtc ttgatggttg atcaaatttt gacaacattt cagcattctt aaacatcttt 176

tcattatttt ttatttacaa agagtagtaa ttcaagcacc ataagaaaca ctgatcaata 236

gtttcttgca agttcttgaa cacttaataa gcagaggggt acttttaaat attcagcatt 296

tgtttgataa tctcaggtgt tttggacttg ctatatgtac ctgatgacac cgctttagtt 356

tcaactagga tatggcgcta aatgggggaa aattgataaa gtcgagtagc aaaaatgatt 416

aggattttaa cgtggtgttt ctccttttct ctctcaagtt cattgtggtg tgccatctat 476

agaaatgtct cgggttgtac tttttctatg gaaatgcagg cgtcgtttca gagtttgttc 536

tctgcttctc tcaatagtca attcagataa gccactttca ctgcaacctt gactgctact 596

cttggacttc aaattctagt cctctttgtc tttgtatcat tcttcaattt ttccaattga 656

tgatgctgat tttgaaaaac tcctctttgc acccgaag cca atg gct ctg gat caa 712

Pro Met Ala Leu Asp Gln

5 10

gta ttg tca gac agg gat agt gag gat gaa gtg gat gat gat att gct 760

Val Leu Ser Asp Arg Asp Ser Glu Asp Glu Val Asp Asp Asp Ile Ala

15 20 25

gct ctt gaa gat aga agg gtacgtttgg ttattttcca aattttttga gttgcttg 816

Ala Leu Glu Asp Arg Arg

30

cgtgattaac aatttttgat ctagtaatgg ttcttgcttc tagccaagtc tttgaatttc 876

taatgtaata gttatctttt tcttgagtgc attttgctaa ctaaaccgtg tatggtacct 936

tgccttgtgc tgcag atg ctt gat gat ttt gtg gat gta agc aaa gac gaa 987

Met Leu Asp Asp Phe Val Asp Val Ser Lys Asp Glu

35 40

aaa cac cta atg cat cta tgg aac tca ttt gta aaa aag caa ag gtagac 1037

Lys His Leu Met His Leu Trp Asn Ser Phe Val Lys Lys Gln Arg

45 50 55

tttgttatgc aattgtcccg tttgtttaat ttctttctcc attgtgaatg cttgcgtagt 1097

gtgctcccga agtatttttg atggcgctta cctgtggttg tttggctttg tgtaatgttt 1157

ccatttttgt gcaccag g gtt ttg gct gat ggt cat gtt ccc tgggcatgcg a 1210

Val Leu Ala Asp Gly His Val Pro

60 65

agtatt 1216