JP2007202425A - Coloration control gene for plant body, and use of the gene - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide genes that control the coloration of a plant body, more specifically, genes that control the coloration of a plant body or its seed, and to provide a method for producing a plant body(transformed plant body) in which either one of the genes is expressed and its seed is colored, furthermore, to provide a method for controlling seed color by modifying the expression of either one of the genes. <P>SOLUTION: It has been succeeded to identify Rc and Rd genes having the function of controlling the coloration of a plant body. By expressing either one of these genes in a plant body, white rice can be modified to e.g. red-colored rice/brown-colored rice. Besides, by this method, a plant body or seeds thereof accumulated with tannins can be created. Rice accumulated with tannins is useful as a functional food. Furthermore, by suppressing the expression of either one of these genes, colored rice can also be modified to white rice. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention utilizes a gene that controls coloring of a plant body or seed thereof, a plant body (transformed plant body) that expresses and colors the gene or a method for producing the seed, and the gene (DNA sequence). The present invention relates to a plant and seed to be selectively bred, and a method for controlling the color of seed by modifying expression of the gene.

Rice seeds, which are said to have red or reddish brown rice, are known to accumulate catechins, tannins, etc., but although the locus on the chromosome has been identified more than 50 years ago, it is still a gene. No level decision has been made.
Prior art document information related to the invention of the present application is shown below.

Kinoshita, T. & Takahashi, M. (1991) J. Fac. Agr. Hokkaido Univ. 65, 1-61. Kinoshita, T. (1995) Rice Genetics Newsletter 12, 9-153. Chen, M. & L. J. Bennetzen (1996) Plant Molecular Biology 32, 999-1001. Nakai, K., Inagaki, Y., Nagata, H., Miyazaki, C., & Iida, S. (1998) Plant Biotechnology 15, 221-225.

  It is an object to provide a gene that controls coloring of a plant body. More specifically, a gene that controls coloring of a plant body or its seed, a method for producing a plant body (transformed plant body) in which the gene is expressed and the seed is colored, and selection using the gene (DNA sequence) It is an object of the present invention to provide a plant body and seed to be bred and a method for controlling the color of the plant body or its seed by modifying the expression of the gene.

  In order to solve the above problems, the present inventors tried to identify Rc and Rd genes involved in red rice pigment coloring of rice.

  Regarding the Rc gene, several candidate genes identified by the present inventors were introduced into Nipponbare using reverse genetic techniques, and a gene having the function of changing the phenotype from white to reddish brown was identified as the causative gene. . From its structural features, it became clear for the first time that the Rc gene is a MYC gene, a transcription factor with a bHLH domain. The function of the gene identified by the present inventors has not been clarified so far, and the present inventors have proved for the first time that the MYC gene is involved in red rice-related pigment expression.

  The region of the locus on the chromosome where the Rc gene is thought to exist is a very wide region (2.3 Mbp), and it is very difficult to identify the causative gene that actually controls the coloration of the plant body It was difficult. That is, in the above region, the number of genes considered to encode a protein having a length of 100 or more amino acids is as high as 200, and it is extremely difficult to identify the causative gene that controls coloration. It is a thing. Identification of the causative gene is accompanied by excessive difficulty even by those skilled in the art, and it can be said that the causal gene was actually identified by the present inventors.

  The Rc (MYC) gene, a gene identified by the present inventors, has so far been known to have no function, and has been found for the first time by the present inventors to have a function of controlling plant coloration. It was done.

  Further, as a result of intensive studies by the present inventors, the Rd gene was found to be a gene DFR. Three types of polymorphisms were recognized in the DFR gene structure. The present inventors made a DFR antibody and analyzed the expression at the protein level, and proved for the first time that it was translated as a plurality of peptides by seed-specific alternative translation initiation.

  Furthermore, the present inventors have found that Rd encodes an enzyme protein (DFR), and the expression related to the coloration is controlled by a regulatory gene MYC which is Rc. Translation of DFR protein is controlled by seed-specific alternative translation initiation. As a result, it was newly observed that rice with full-length DFR has a red seed, rice with a short DFR ORF has a reddish brown color, and when Rc does not function, the seed has a white color. It was issued.

  A more detailed analysis showed that in the rd form, the DFR gene was translated from the downstream by alternative translation initiation, producing 33 kDa and 22 kDa translation products instead of the usual 46 kDa translation products. . Furthermore, the present inventors have found for the first time that the 33 kDa and 22 kDa products translated by the alternative translation initiation mechanism can complement the function of DFR. That is, in addition to the normal full-length DFR protein, it has been found that a translation product having a smaller molecular weight has a function of coloring a plant body or a function of accumulating tannins.

  As described above, the present inventors have succeeded in isolating a gene that controls the coloration of a plant body or its seeds, thereby completing the present invention. By using this gene, it is possible to control the coloring of the plant body, particularly the seed.

The present invention relates to a gene that controls coloration of a plant or its seed, a method for producing a plant (transformed plant) in which the gene is expressed and the seed is colored, and selection using the gene (DNA sequence) Plants and seeds to be bred, and further to a method for controlling the color of plants, particularly seeds, by modifying the expression of the gene, more specifically,
[1] Any of the following (a) to (e), which encodes a plant-derived protein having a function of coloring a plant body or its seed or a function of accumulating tannins in the plant body or its seed. DNA,
(A) DNA encoding a protein comprising the amino acid sequence of SEQ ID NO: 3 or 6-8.
(B) DNA comprising the coding region of the nucleotide sequence set forth in SEQ ID NO: 1 or 4.
(C) DNA comprising the nucleotide sequence set forth in SEQ ID NO: 2 or 5
(D) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 3 or 6-8.
(E) A DNA that hybridizes under stringent conditions with a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1 or 4.
[2] The DNA according to [1], wherein the plant is a monocotyledonous plant,
[3] The DNA according to [2], wherein the monocotyledonous plant is rice,
[4] a protein encoded by the DNA according to any one of [1] to [3],
[5] A vector comprising the DNA according to any one of [1] to [3],
[6] A host cell into which the vector according to [5] has been introduced,
[7] A plant cell into which the vector according to [5] has been introduced,
[8] A transformed plant comprising the plant cell according to [7],
[9] A transformed plant that is a descendant or clone of the transformed plant according to [8],
[10] A propagation material for the transformed plant according to [8] or [9],
[11] The method for producing a protein according to [4], comprising culturing the host cell according to [6] and recovering the recombinant protein from the cell or a culture supernatant thereof,
[12] an antibody that binds to the protein of [4],
[13] A polynucleotide comprising at least 15 consecutive bases complementary to the base sequence of the DNA according to any one of [1] to [3] or a complementary sequence thereof,
[14] A plant coloring agent containing any of the following (a) to (c) as an active ingredient,
(A) DNA according to any one of [1] to [3]
(B) The protein according to [4] (c) The vector according to [5] [15] A tannin accumulating agent containing any of the following (a) to (c) as an active ingredient,
(A) DNA according to any one of [1] to [3]
(B) the protein according to [4] (c) the vector according to [5] [16] a plant decolorizing agent containing any of the following (a) to (c) as an active ingredient,
(A) an antisense nucleic acid for the transcription product of the DNA according to any one of [1] to [3] or a part thereof; and the DNA transcription product according to any one of [1] to [3] A nucleic acid having a cleaving ribozyme activity (c) a nucleic acid having an inhibitory action by the RNAi effect on the expression of the DNA according to any one of [1] to [3] [17] any one of [1] to [3] A method for producing a transformed plant, comprising the step of introducing the DNA according to claim 5 or the vector according to [5] into a plant cell and regenerating the plant from the plant cell,
[18] The method includes the step of expressing the DNA according to any one of [1] to [3] in cells of the plant body, coloring the plant body or its seed, or accumulating tannins in the plant body or its seed. How to
[19] A method for producing a colored or tannin-accumulated plant or a seed thereof, comprising the step of expressing the DNA according to any one of [1] to [3] in a cell of the plant,
[20] The method according to [18] or [19], comprising the step of introducing the DNA according to any one of [1] to [3] or the vector according to [5] into a plant cell,
[21] The method according to [18] or [19], comprising the following steps (a) and (b):
(A) including a step of crossing with a plant having the DNA according to any one of [1] to [3],
(B) A step of selecting a plant variant having the DNA [22] A method of breeding a plant having a colored seed or a seed having accumulated tannins, comprising the following steps (a) to (d): Breeding method,
(A) A step of crossing plant A with another plant B having the DNA of any one of [1] to [3] to produce F1 (b) A step of crossing F1 and plant A c) a step of selecting a plant having the DNA (d) a step of crossing the plant selected in the step (c) with the plant A [23] In the step (c), [1] to [1] The method according to [22], wherein the DNA sequence according to any one of [3] or a DNA marker present in its peripheral sequence is used for selection.
[24] A step of suppressing the expression of a gene encoding a protein that is endogenous to a plant and has a function of coloring a plant or its seed, or a function of accumulating tannins in a plant or its seed. A method for producing a decolorized plant or its seed,
[25] The method according to any one of [17] to [24], wherein the plant is rice.
[26] A plant obtained by the method according to any one of [18] to [25], or a seed thereof,
[27] An artificially produced plant or seed thereof, wherein the plant or seed is characterized by having the DNA of any one of [1] to [3] and being colored,
[28] An artificially produced plant or a seed thereof, wherein the plant has the DNA of any one of [1] to [3] and tannins are accumulated, or Its seeds,
[29] Artificial suppression of expression of a gene encoding a protein that is endogenous to a plant and has a function of coloring the plant body or its seed or a function of accumulating tannins in the plant body or its seed A plant or its seed characterized by being decolorized,
[30] The plant according to any one of [27] to [29] or a seed thereof, wherein the plant is rice.

  By the method of the present invention, a technique for artificially coloring a plant or its seed (for example, rice) by expressing an Rc or Rd gene in the plant body has been established. In order to respond to social concerns such as genetically modified organics (GMO) mixed into general seeds, GMO rice can be handled by artificially coloring it. One of the advantages of the present invention is that GMO can be monitored, for example, by coloring rice peels. If necessary, it is also possible to finally remove the colored portion by using polished rice.

  In addition, good-tasting rice can be bred into colored rice in a few years by the breeding method using DNA markers in the present invention. It is possible to breed good-tasting colored rice or functional good-tasting rice lines such as “Koshihikari catechin”, “catechin human mebore”, and “catechin mangetsumochi” in a very short time. Although the taste is completely undeveloped colored rice, good taste colored rice can be developed by using the technique of the present invention. Since the plant produced by the method of the present invention contains catechin and tannin having a high antioxidant function, for example, it is possible to efficiently produce rice having added value as functional rice.

  The present inventors have identified a gene having a function of coloring a plant body or a function of accumulating tannins in a plant body.

  A preferred embodiment of the gene of the present invention includes rice Rc gene or Rd gene. The protein encoded by the Rc gene is a proanthocyanidin synthesis system controlling factor and has a function as a transcriptional control factor. The Rc gene may be referred to as “MYC gene”, “Rc-MYC gene”, “Myc related protein gene” or the like.

  On the other hand, the Rd gene may be referred to as “DFR gene” or “Dihydroflavonol 4-reductase gene”. The expression was regulated by the transcription factor Rc (MYC). In addition, the present inventors have found that translation of Rd (DFR) protein is controlled by Alternative translation initiation. In plants with full length DFR (eg, rice), the seeds are red, in plants with a short DFR ORF (eg, rice), the seeds are reddish brown, and when Rd does not function, the seeds are white Presents.

  As shown in the examples described later, the present inventors have found for the first time that rd-type DFR is translated from downstream ATG by Alternative translation initiation, and that this translation product has a function of complementing the function of DFR. It was done. Therefore, in the present invention, the Rd gene is not limited to DNA encoding the full-length DFR protein, and includes DNA encoding a product translated from ATG downstream of DFR that can complement the function of DFR.

  For example, the Rd (DFR) protein of the present invention includes proteins corresponding to 46 kDa (SEQ ID NO: 6), 33 kDa (SEQ ID NO: 7), and 22 kDa (SEQ ID NO: 8) shown in FIG. It is done. The proteins corresponding to the 33 kDa and 22 kDa are considered to be translated from the Rd (DFR) gene by the Alternative translation initiation mechanism.

  In addition to the above proteins, DFR protein mutants or partial fragment peptides of the DFR protein that can complement the DFR function are also included in the Rd (DFR) protein of the present invention.

  The Rc and Rd proteins of the present invention are preferably rice-derived proteins, but the plant species from which they are derived is not particularly limited, and proteins equivalent to Rc or Rd in plants other than rice (Rc or Rd homologs) Orthologs etc.) are also included in the “Rc protein” or “Rd protein” in the present invention. For example, the present invention can be implemented as long as it is a plant having a protein corresponding to rice Rc (MYC) or Rd (DFR), or a plant in which the rice Rc or Rd protein can substantially function.

  In the present invention, the “plant” is not particularly limited, but is preferably a monocotyledonous plant, more preferably a gramineous plant. Specific examples of the plant in the present invention include rice, corn, wheat, barley and the like. (These Rc and Rd proteins and the gene encoding the protein may be referred to as “the protein of the present invention” and “the gene of the present invention”, respectively, in the present specification.)

  The genomic DNA sequence of the Rc (MYC) gene is described in SEQ ID NO: 1, the cDNA sequence of the Rc (MYC) gene is described in SEQ ID NO: 2, and the amino acid sequence of the protein encoded by the DNA is described in SEQ ID NO: 3. Further, the genomic DNA sequence of the Rd (DFR) gene is shown in SEQ ID NO: 4, the cDNA sequence of the Rd (DFR) gene is shown in SEQ ID NO: 5, and the amino acid sequence of the protein encoded by the DNA is shown in SEQ ID NO: 6. To do. (The GenBank accession number of rice Rc (MYC) is AB247503. The GenBank accession number of rice Rd (DFR) is AB003496.)

  However, the Rc or Rd gene in the present invention is not necessarily limited to DNA having a sequence specifically described in the sequence listing. In addition, the Rc or Rd protein in the present invention is not necessarily limited to a protein having an amino acid sequence specifically described in the sequence listing.

  Even proteins other than the above have high homology (usually 70% or more, preferably 80% or more, more preferably 90% or more, most preferably 95% or more) with the sequences described in the sequence listing, for example. A protein having a function of the protein of the present invention (for example, a function of coloring seeds or a function of accumulating tannins in seeds) is included in the protein of the present invention.

  The protein is, for example, a protein comprising an amino acid sequence in which one or more amino acids are added, deleted, substituted, or inserted in the amino acid sequence of SEQ ID NO: 3 or 6-8, The number of amino acids to be changed is within 30 amino acids, preferably within 10 amino acids, more preferably within 5 amino acids, and most preferably within 3 amino acids.

  The “Rc gene” or “Rd gene” in the present invention includes, for example, an endogenous gene (such as rice Rc or Rd gene) corresponding to a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 2 or 5. Homologs, etc.).

  In addition, endogenous DNA of other plants corresponding to the DNA consisting of the base sequence described in SEQ ID NO: 1, 2, 4 or 5 is generally described in SEQ ID NO: 1, 2, 4 or 5. High homology with DNA. High homology means 50% or more, preferably 70% or more, more preferably 80% or more, more preferably 90% or more (for example, 95% or more, or 96%, 97%, 98% or 99% or more). ) Homology. This homology is determined by the mBLAST algorithm (Altschul et al. (1990) Proc. Natl. Acad. Sci. USA 87: 2264-8; Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-7 ) Can be determined. In addition, when isolated from the living body, the DNA is considered to hybridize with the DNA described in SEQ ID NO: 1, 2, 4 or 5 under stringent conditions. Here, as “stringent conditions”, for example, “2 × SSC, 0.1% SDS, 50 ° C.”, “2 × SSC, 0.1% SDS, 42 ° C.”, “1 × SSC, 0.1% SDS, 37 ° C.” More stringent conditions include “2 × SSC, 0.1% SDS, 65 ° C.”, “0.5 × SSC, 0.1% SDS, 42 ° C.” and “0.2 × SSC, 0.1% SDS, 65 ° C.” Can do. Those skilled in the art can appropriately obtain an endogenous gene corresponding to the gene of the present invention in other plants based on the base sequence of the gene of the present invention. In the present specification, proteins (genes) corresponding to Rc proteins (genes) or Rd proteins (genes) in plants other than rice, or proteins (genes) functionally equivalent to these proteins, It may be described as “protein (gene) of the present invention”.

  The protein of the present invention can be prepared not only as a natural protein but also as a recombinant protein using a gene recombination technique. Natural proteins can be prepared, for example, by a method using affinity chromatography using an antibody against the protein of the present invention to an extract of a cell (tissue) that is considered to express the protein of the present invention. is there. On the other hand, a recombinant protein can be prepared by culturing cells transformed with a DNA encoding the protein of the present invention.

As a preferred embodiment of the present invention, the following (a) to (e) encoding a plant-derived protein having a function of coloring a plant body or its seed or a function of accumulating tannins in the plant body or its seed. A DNA according to any of the above is provided.
(A) DNA encoding a protein comprising the amino acid sequence of SEQ ID NO: 3 or 6-8
(B) DNA containing the coding region of the base sequence described in SEQ ID NO: 1 or 4
(C) DNA comprising the nucleotide sequence set forth in SEQ ID NO: 2 or 5
(D) DNA encoding a protein consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 3 or 6-8
(E) DNA that hybridizes under stringent conditions with DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1 or 4

In addition, a protein encoded by the above DNA is also included in the present invention.
In the present invention, “plant seed” refers to “rice”, “brown rice”, etc., for example, when the plant is rice.

  In the present invention, “coloring” usually means changing an original color to another color in a plant or its seed. As an example, it refers to coloring the whole or part of the seed into red, reddish brown, brown, purple black or the like. More specifically, when the plant is rice, it means coloring white rice (white rice) to red rice (red rice), reddish brown rice, brown rice, purple black rice or the like. (As described on the left, colored rice other than white rice may be referred to as “colored rice” in the present invention.)

  Further, in the present invention, “controlling the coloring” means changing the color of the whole plant or its seeds or a part thereof to another color as described above. For example, when the plant is rice, as described above, it is said that white rice is changed to colored rice such as red rice. On the other hand, when changing colored rice to white rice, It is included in “control”.

  The part of the plant body in which coloring is controlled in the present invention is not particularly limited, and examples thereof include seeds, leaves, internodes, stems, and gills (fir).

  When the plant in the present invention is rice, for example, the seed coat and / or pericarp of rice seed (rice) is colored. In addition, when the plant in the present invention is wheat, barley, corn or the like, the seed peel is colored.

  The coloring part of the plant in the present invention is not necessarily limited to the above-mentioned part, and it is also possible to color internodes of plants, endosperm of seeds, and the like.

  “Tannin” is a general term for aromatic compounds having a large number of phenolic hydroxyl groups widely distributed in the plant kingdom, and is referred to as “tannic acid” or “gallotannin”. Also called “gallotannic acid”.

  The “tannins” in the present invention include, for example, “tannin”, “catechin”, “anthocyanin”, “proanthocyanidin” and the like. The catechin is also referred to as “3,3 ′, 4 ′, 5,7-pentahydroxyflavan”. “Catechin” also includes compounds belonging to so-called “catechins” such as epicatechin. In addition, substances produced by the reaction of tannins with various compounds are also included in the “tannins” in the present invention. The “anthocyanin” is a purple pigment, and the “proanthocyanidins” are considered to be reddish brown or red pigments.

  The brown rice of the red rice variety “Koi” contains 0.29 g of tannin, 1.6 mg of catechin, and 0.09 g of anthocyan in 100 g. Therefore, when tannins are accumulated in white rice by the method of the present invention, examples of the amount (content) of tannins accumulated in rice include the above amounts.

  In the present invention, “accumulating tannins” usually means generating and accumulating tannins in a site where tannins originally do not exist in the plant, or tannins in sites where tannins exist in the plant. It means to increase the amount (content).

  The protein in this specification means a polymer composed of a plurality of amino acids, and the length of the amino acids is not particularly limited. Therefore, the protein of the present invention includes so-called “polypeptides” and “oligopeptides”. The proteins of the present invention include both those that are not modified from the naturally occurring state and those that are modified. Modifications include acetylation, acylation, ADP-ribosylation, amidation, flavin covalent bond, heme moiety covalent bond, nucleotide or nucleotide derivative covalent bond, lipid or lipid derivative covalent bond, phosphatidylinositol covalent bond , Crosslinking, cyclization, disulfide bond formation, demethylation, covalent bridge formation, cystine formation, pyroglutamate formation, formylation, γ-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, Examples include methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer RNA-mediated addition of amino acids to proteins, ubiquitination, and the like.

  The polypeptide of the present invention can be produced by a general chemical synthesis method according to its amino acid sequence, and this method includes peptide synthesis methods by a normal liquid phase method and a solid phase method. More specifically, the peptide synthesis method includes a stepwise elongation method in which each amino acid is sequentially synthesized one by one based on the amino acid sequence information to extend the chain, and a fragment consisting of several amino acids is synthesized in advance. Then, a fragment condensation method in which each fragment is subjected to a coupling reaction is included, and any method may be used for the synthesis of the protein of the present invention.

  The condensation method used in such a peptide synthesis method can also be performed according to various methods. Specific examples thereof include an azide method, a mixed acid anhydride method, a DCC method, an active ester method, an oxidation-reduction method, a DPPA (diphenylphosphoryl azide) method, and a Woodward method.

  As the solvent that can be used in these various methods, those generally used can be appropriately used. Examples thereof include dimethylformamide (DMF), dimethyl sulfoxide (DMSO), hexaphosphoroamide, dioxane, tetrahydrofuran (THF), ethyl acetate and the like, and mixed solvents thereof. In the peptide synthesis reaction, amino acids that are not involved in the reaction and carboxyl groups in the peptide are generally esterified, for example, lower alkyl esters such as methyl ester, ethyl ester, tertiary butyl ester, such as benzyl ester, P- It can be protected as methoxybenzyl ester, P-nitrobenzyl ester aralkyl ester and the like. An amino acid having a functional group in the side chain, for example, a hydroxyl group of Tyr may be protected with an acetyl group, a benzyl group, a benzyloxycarbonyl group, a tertiary butyl group, or the like, but such protection is not necessarily essential. Further, for example, the guanidino group of Arg is a suitable nitro group, tosyl group, 2-methoxybenzenesulfonyl group, methicylene-2-sulfonyl group, benzyloxycarbonyl group, isobornyloxycarbonyl group, adamantyloxycarbonyl group, etc. It can be protected by a protecting group.

  The protein of the present invention that can be obtained as described above can be obtained by, for example, ion exchange resin, partition chromatography, gel chromatography, affinity chromatography, high performance liquid chromatography (HPLC), countercurrent distribution method according to a conventional method. Purification can be appropriately performed according to a method widely used in the field of peptide chemistry.

  The protein of the present invention can be synthesized by, for example, synthesizing the polypeptide shown in SEQ ID NO: 3 or 6 to 8 or the DNA nucleic acid molecule shown in SEQ ID NO: 2 or 5 and then introducing it into a suitable expression vector. It can also be obtained by a genetic engineering technique for expression in cells.

  The protein of the present invention includes, for example, a protein (polypeptide) functionally equivalent to the Rc (MYC) or Rd (DFR) protein. Here, “functionally equivalent” means that the target protein has the same (equivalent) biological or biochemical function (activity) as the Rc (MYC) or Rd (DFR) protein. Examples of such a function include a function of coloring a plant or its seed, a function of accumulating tannins in a plant or its seed, and the like.

  Whether a certain DNA encodes a protein having a function of coloring a plant body or a function of accumulating tannins can be evaluated as follows. The most common method is a method of cultivating a plant into which the DNA has been introduced and evaluating the color of the plant body or the amount of tannins contained in the plant body.

  Whether the target protein (polypeptide) has the same biological or biochemical function (activity) as the Rc (MYC) or Rd (DFR) protein is expressed, for example, by expressing the protein It is also possible to appropriately evaluate by measuring the amount of tannins in the plant or the seeds of the plant.

  A method well known to those skilled in the art for preparing a polypeptide functionally equivalent to a certain polypeptide includes, for example, a method of introducing a mutation into an amino acid sequence in the polypeptide. Specifically, a person skilled in the art can perform site-directed mutagenesis (Hashimoto-Gotoh, T. et al. (1995) Gene 152, 271-275, Zoller, MJ, and Smith, M. (1983) Methods Enzymol. 100, 468-500, Kramer, W. et al. (1984) Nucleic Acids Res. 12, 9441-9456, Kramer W, and Fritz HJ (1987) Methods. Enzymol. 154, 350-367, Kunkel, TA (1985) ) Proc Natl Acad Sci USA. 82, 488-492, Kunkel (1988) Methods Enzymol. 85, 2763-2766) and the like, by appropriately introducing mutation into the amino acid sequence described in SEQ ID NO: 4 A polypeptide functionally equivalent to the polypeptide can be prepared. In addition, amino acid mutations in the polypeptide may occur naturally. Thus, one or more amino acid sequences in the amino acid sequence of the Rc or Rd protein (SEQ ID NO: 3 or 6-8) identified by the present inventors, whether artificial or naturally occurring, A polypeptide having a mutated amino acid sequence and functionally equivalent to the polypeptide is included in the protein (polypeptide) of the present invention.

  The number of amino acids to be mutated in the mutant is not limited as long as the function of the protein of the present invention is maintained, but is usually within 15 amino acids, preferably within 10 amino acids, more preferably within 5 amino acids. More preferably 1 to 4 amino acids.

  The amino acid residue to be mutated is preferably mutated to another amino acid that preserves the properties of the amino acid side chain. For example, amino acid side chain properties include hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), amino acids having aliphatic side chains (G, A, V, L, I, P), amino acids having hydroxyl group-containing side chains (S, T, Y), sulfur atom-containing side chains Amino acids with amino acids (C, M), amino acids with carboxylic acid and amide-containing side chains (D, N, E, Q), amino groups with base-containing side chains (R, K, H), aromatic-containing side chains (H, F, Y, W) can be mentioned (all parentheses indicate single letter amino acids).

  It is possible that a polypeptide having an amino acid sequence modified by deletion, addition and / or substitution by another amino acid of one or more amino acid residues to a certain amino acid sequence can maintain its biological function (activity). Already known (Mark, DF et al., Proc. Natl. Acad. Sci. USA (1984) 81, 5662-5666, Zoller, MJ & Smith, M. Nucleic Acids Research (1982) 10, 6487-6500 Wang, A. et al., Science 224, 1431-1433, Dalbadie-McFarland, G. et al., Proc. Natl. Acad. Sci. USA (1982) 79, 6409-6413).

  In the case where a specific amino acid sequence (for example, SEQ ID NO: 3 or 6 to 8) is disclosed, those skilled in the art will obtain a polypeptide comprising a sequence in which amino acids are appropriately modified based on these amino acid sequences. It is possible to prepare and evaluate whether or not the polypeptide has the above-described function, and appropriately select the protein (polypeptide) of the present invention.

  It has been clarified that the Rd protein in the present invention can complement the functions of the protein of the present invention even when its N-terminal region is missing. Therefore, as a modified form of the Rd protein, for example, a modified form (variant, partial peptide, etc.) of the Rd protein containing the amino acid region represented by SEQ ID NO: 7 or 8 and having a mutation or deletion in the N-terminal region. It can be illustrated.

  Proteins in which a plurality of amino acid residues are added to the amino acid sequence of the protein of the present invention include fusion polypeptides containing these proteins. A fusion polypeptide is a fusion of these polypeptides with other peptides or polypeptides. A method for producing a fusion polypeptide includes a DNA (eg, SEQ ID NO: 2 or 5) encoding a protein of the present invention (eg, SEQ ID NO: 3 or 6-8) and a DNA encoding another peptide or polypeptide. Can be linked to each other in a frame, introduced into an expression vector, and expressed in a host. Techniques known to those skilled in the art can be used. Other peptides or polypeptides that are subjected to fusion with the protein of the present invention are not particularly limited.

  Examples of other peptides that are subjected to fusion with the polypeptide of the present invention include GST (glutathione-S-transferase), immunoglobulin constant region, β-galactosidase, and MBP (maltose binding protein). Preparing a fusion polypeptide by fusing a commercially available polynucleotide encoding these peptides or polypeptides with a polynucleotide encoding the polypeptide of the present invention and expressing the fusion polynucleotide prepared thereby. Can do.

  Other methods well known to those skilled in the art for preparing polypeptides functionally equivalent to a polypeptide include hybridization techniques (Sambrook, J et al., Molecular Cloning 2nd ed., 9.47-9.58, Cold Spring Harbor Lab. Press, 1989). That is, those skilled in the art can use a DNA sample derived from the same or different plant based on a DNA encoding the protein of the present invention (base sequence described in SEQ ID NO: 2 or 5) or a part thereof. It is usually possible to isolate highly homologous DNA and isolate a polypeptide functionally equivalent to the protein of the present invention from the DNA.

  The present invention includes proteins encoded by DNA that hybridizes with DNA encoding the protein of the present invention and functionally equivalent to the protein of the present invention. Examples of such proteins include rice or other plant homologs (for example, proteins derived from plants such as corn, wheat, barley, etc.).

  Those skilled in the art can appropriately select hybridization conditions for isolating a DNA encoding a protein functionally equivalent to the protein of the present invention. Examples of hybridization conditions include low stringency conditions. Low stringent conditions are, for example, conditions of 42 ° C., 0.1 × SSC, and 0.1% SDS, and preferably 50 ° C., 0.1 × SSC, and 0.1% SDS in washing after hybridization. More preferable hybridization conditions include highly stringent conditions. Highly stringent conditions are, for example, conditions of 65 ° C., 5 × SSC and 0.1% SDS. Under these conditions, it can be expected that DNA having high homology can be efficiently obtained as the temperature is increased. However, a plurality of factors such as temperature and salt concentration can be considered as factors affecting the stringency of hybridization, and those skilled in the art can realize the same stringency by appropriately selecting these factors. .

  In addition, DNA encoding the protein of the present invention can be obtained by using gene amplification technology (PCR) (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons Section 6.1-6.4) instead of hybridization. A primer is designed based on a part of (for example, SEQ ID NO: 2 or 5), a DNA fragment having high homology with DNA encoding the protein identified by the present inventors is isolated, and based on the DNA It is also possible to obtain a protein functionally equivalent to the protein identified by the present inventors.

  The protein of the invention may be in the form of a “mature” polypeptide or may be part of a larger polypeptide, such as a fusion polypeptide. The protein of the present invention may contain a leader sequence, a pro sequence, a sequence useful for purification, such as multiple histidine residues, or an additional sequence ensuring stability during recombinant production.

  The protein functionally equivalent to the protein of the present invention encoded by DNA isolated by these hybridization techniques and gene amplification techniques is usually the protein of the present invention (for example, SEQ ID NO: 3 or 6 to 8). High homology in amino acid sequence. The protein of the present invention includes a protein that is functionally equivalent to the Rc or Rd protein and has a high homology with the amino acid sequence of the protein. High homology is usually at least 50% identity, preferably 75% identity, more preferably 85% identity, more preferably 95% (eg 96%) at the amino acid level. Above, 97% or higher, 98% or higher, 99% or higher). To determine protein homology, algorithms described in the literature (Wilbur, W. J. and Lipman, D. J. Proc. Natl. Acad. Sci. USA (1983) 80, 726-730) may be used.

The identity of the amino acid sequence is determined by, for example, the algorithm BLAST by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990, Proc. Natl. Acad. Sci. USA 90: 5873-5877, 1993). Can be determined by. Based on this algorithm, a program called BLASTX has been developed (Altschul et al. J. Mol. Biol. 215: 403-410, 1990). When the amino acid sequence is analyzed by BLASTX, the parameters are, for example, score = 50 and wordlength = 3. When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific methods for these analysis methods are known ( http://www.ncbi.nlm.nih.gov .).

  The protein of the present invention can be prepared as a recombinant polypeptide or a natural polypeptide by methods known to those skilled in the art. In the case of a recombinant polypeptide, for example, a DNA encoding the protein of the present invention (for example, the DNA described in SEQ ID NO: 2 or 5) is incorporated into an appropriate expression vector and introduced into an appropriate host cell. The obtained transformant is recovered and an extract is obtained, followed by chromatography such as ion exchange, reverse phase, gel filtration, or affinity chromatography in which an antibody against the protein of the present invention is immobilized on the column, or Furthermore, it is possible to purify and prepare by combining a plurality of these columns.

  In addition, when the protein of the present invention is expressed in a host cell (for example, plant cell or microbial cell) as a fusion polypeptide with glutathione S-transferase protein or as a recombinant polypeptide to which a plurality of histidines are added. The expressed recombinant polypeptide can be purified using a glutathione column or a nickel column. After purification of the fusion polypeptide, if necessary, a region other than the target polypeptide in the fusion polypeptide can be cleaved with thrombin or factor Xa and removed.

  In the case of a natural protein, an affinity column in which an antibody having an affinity for the protein of the present invention is bound to an extract of a tissue or a cell expressing the protein of the present invention is applied to a method well known to those skilled in the art. And can be isolated by purification. The antibody may be a polyclonal antibody or a monoclonal antibody.

  In addition, the protein of the present invention can be used, for example, for production of an antibody that recognizes the protein of the present invention.

  The DNA of the present invention may be in any form as long as it can encode the protein of the present invention. That is, it does not matter whether it is cDNA synthesized from mRNA, genomic DNA, or chemically synthesized DNA. Moreover, as long as it can code the protein of this invention, DNA which has the arbitrary base sequences based on the degeneracy of a genetic code is contained.

  The DNA of the present invention can be prepared by methods known to those skilled in the art. For example, a cDNA library is prepared from cells expressing the protein of the present invention, and hybridization is carried out using a part of the DNA of the present invention (for example, the nucleotide sequence described in SEQ ID NO: 2 or 5) as a probe. Can be prepared. The cDNA library may be prepared, for example, by the method described in the literature (Sambrook, J. et al., Molecular Cloning, Cold Spring Harbor Laboratory Press (1989)), or a commercially available DNA library may be used. Good. In addition, RNA is prepared from cells expressing the protein of the present invention, cDNA is synthesized by reverse transcriptase, and then oligos based on the DNA of the present invention (for example, the nucleotide sequence described in SEQ ID NO: 2 or 5) are synthesized. It is also possible to synthesize DNA, perform PCR reaction using it as a primer, and amplify the cDNA encoding the protein of the present invention.

  Moreover, by determining the base sequence of the obtained cDNA, the translation region encoded by the cDNA can be determined, and the amino acid sequence of the protein of the present invention can be obtained. In addition, genomic DNA can be isolated by screening a genomic DNA library using the obtained cDNA as a probe.

  Specifically, it may be performed as follows. First, mRNA is isolated from cells, tissues, and organs that express the protein of the present invention. Isolation of mRNA is performed by a known method such as guanidine ultracentrifugation (Chirgwin, JM et al., Biochemistry (1979) 18, 5294-5299), AGPC (Chomczynski, P. and Sacchi, N., Anal. Total RNA is prepared by Biochem. (1987) 162, 156-159) etc., and mRNA is purified from the total RNA using mRNA Purification Kit (Pharmacia) etc. Alternatively, mRNA can be directly prepared by using QuickPrep mRNA Purification Kit (Pharmacia).

  CDNA is synthesized from the obtained mRNA using reverse transcriptase. cDNA synthesis can also be performed using AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (Seikagaku Corporation). In addition, using the primers described in this specification, 5′-Ampli FINDER RACE Kit (manufactured by Clontech) and 5′-RACE method using polymerase chain reaction (PCR) (Frohman, MA et al.) al., Proc. Natl. Acad. Sci. USA (1988) 85, 8998-9002; Belyavsky, A. et al., Nucleic Acids Res. (1989) 17, 2919-2932). It can be carried out. A target DNA fragment is prepared from the obtained PCR product and ligated with vector DNA. Further, a recombinant vector is prepared from this, introduced into Escherichia coli, etc., and colonies are selected to prepare a desired recombinant vector. The base sequence of the target DNA can be confirmed by a known method, for example, the dideoxynucleotide chain termination method.

  In the preparation of the DNA of the present invention, a base sequence with higher expression efficiency can be designed in consideration of the codon usage frequency of the host used for expression (Grantham, R. et al., Nucelic Acids Research ( 1981) 9, r43-74). Moreover, the DNA of the present invention can be modified by a commercially available kit or a known method. Examples of modifications include digestion with restriction enzymes, insertion of synthetic oligonucleotides and appropriate DNA fragments, addition of linkers, insertion of start codon (ATG) and / or stop codon (TAA, TGA, or TAG). .

  The present invention also provides a vector into which the DNA of the present invention is inserted. The vector of the present invention includes a vector for expressing the DNA of the present invention in a plant cell in addition to the above-mentioned vector used for production of a recombinant protein in order to produce a transformed plant body. Such a vector preferably contains a promoter sequence that can be transcribed in plant cells and a terminator sequence including a polyadenylation site necessary for stabilization of the transcript. The vector used for the transformation of plant cells is not particularly limited as long as the inserted gene can be expressed in the cells. For example, using a vector having a promoter (eg, cauliflower mosaic virus 35S promoter) for constitutive gene expression in plant cells or a vector having a promoter that is inducibly activated by external stimuli Is also possible. The “plant cell” includes various forms of plant cells, such as suspension culture cells, protoplasts, leaf sections, callus and the like.

  The vector of the present invention may contain a promoter for constitutively or inducibly expressing the protein of the present invention. Examples of promoters for constant expression include cauliflower mosaic virus 35S promoter, rice actin promoter, corn ubiquitin promoter, and the like.

  In addition, promoters for inducible expression include promoters that are known to be expressed by external factors such as bacterial / virus infection and invasion, low temperature, high temperature, drying, ultraviolet irradiation, and application of specific compounds. Etc. Examples of such promoters include rice chitinase gene promoters expressed by bacterial and viral infection and invasion, tobacco PR protein gene promoters, rice "lip19" gene promoters induced by low temperatures, and induced by high temperatures. Promoters of rice “hsp80” and “hsp72” genes, Arabidopsis thaliana “rab16” gene promoter, Parsley chalcone synthase gene promoter induced by UV irradiation, under anaerobic conditions Examples thereof include a promoter of an induced corn alcohol dehydrogenase gene. The rice chitinase gene promoter and tobacco PR protein gene promoter are also induced by specific compounds such as salicylic acid, and “rab16” is also induced by spraying the plant hormone abscisic acid.

  Those skilled in the art can appropriately prepare a vector having a desired DNA by a general genetic engineering technique. Usually, various commercially available vectors can be used.

  The vector of the present invention is also useful for retaining the DNA of the present invention in a host cell or for expressing the protein of the present invention.

  The DNA in the present invention is usually carried (inserted) into an appropriate vector and introduced into a host cell. The vector is not particularly limited as long as it can stably hold the inserted DNA. For example, if Escherichia coli is used as a host, a pBluescript vector (Stratagene) is preferred as a cloning vector, but commercially available. Various vectors can be used. An expression vector is particularly useful when a vector is used for the purpose of producing the protein of the present invention. The expression vector is not particularly limited as long as it is a vector that expresses the polypeptide in vitro, in E. coli, in cultured cells, or in individual plants. For example, in the case of in vitro expression, pBEST vector (manufactured by Promega), E. coli PET vector (manufactured by Invitrogen), pME18S-FL3 vector (GenBank Accession No. AB009864) for cultured cells, pME18S vector (Mol Cell Biol. 8: 466-472 (1988)) for organisms, etc. Can be illustrated. The nucleic acid of the present invention can be inserted into the vector by a conventional method, for example, by a ligase reaction using a restriction enzyme site.

  There is no restriction | limiting in particular as said host cell, According to the objective, various host cells are used. Examples of cells for expressing the protein of the present invention include bacterial cells (eg, Streptococcus, Staphylococcus, E. coli, Streptomyces, Bacillus subtilis), insect cells (eg, Drosophila S2, Spodoptera SF9), animal cells ( Examples: CHO, COS, HeLa, C127, 3T3, BHK, HEK293, Bowes melanoma cells) and plant cells. Vector introduction into host cells can be performed by, for example, calcium phosphate precipitation, electric pulse perforation (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 9.1-9.9), lipofection (GIBCO -BRL) and a known method such as a microinjection method.

  In order to secrete the protein expressed in the host cell into the lumen of the endoplasmic reticulum, into the periplasmic space, or into the extracellular environment, an appropriate secretion signal can be incorporated into the protein of interest. These signals may be endogenous to the protein of interest or they may be heterologous signals.

  When the protein of the present invention is secreted into the medium, the medium is recovered in the production method. When the protein of the present invention is produced intracellularly, the cell is first lysed and then the protein is recovered.

  To recover and purify the protein of the invention from recombinant cell culture, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyl Known methods including apatite chromatography and lectin chromatography can be used.

  In addition, as a method for expressing the DNA of the present invention in a living body of a plant, the DNA of the present invention is incorporated into an appropriate vector, for example, by electroporation method, Agrobacterium method, liposome method, cationic liposome method, etc. Examples thereof include a method of introducing into a living body. General genetic manipulations such as insertion of the DNA of the present invention into a vector can be performed according to conventional methods (Molecular Cloning, 5.61-5.63). Administration into a plant may be an ex vivo method or an in vivo method. In addition, the method for introducing the DNA of the present invention into a plant body preferably includes a method for introducing a gene via Agrobacterium.

  It is also possible to prepare a transformed plant into which the DNA of the present invention has been introduced by the method described later and prepare the protein of the present invention from the plant.

  Moreover, if the recombinant protein obtained as mentioned above is used, the antibody couple | bonded with this can be prepared. For example, a polyclonal antibody can be prepared by immunizing an immunized animal such as a rabbit with the purified protein of the present invention or a partial peptide thereof, collecting blood after a certain period, and removing the blood clot. is there. In addition, the monoclonal antibody is obtained by fusing an antibody-producing cell of an animal immunized with the protein or peptide and a bone tumor cell, and isolating a single clone cell (hybridoma) that produces the target antibody. Can be prepared. The antibody thus obtained can be used for purification and detection of the protein of the present invention. The present invention includes antibodies that bind to the proteins of the present invention. By using these antibodies, it is possible to determine the expression site of the protein of the present invention in a plant body, or to determine whether the plant species express the protein of the present invention.

  When producing a transformed plant body that is colored or accumulated with tannins using the DNA of the present invention, the DNA encoding the protein of the present invention is inserted into an appropriate vector, and this is transformed into plant cells. And transformed plant cells obtained thereby are regenerated. The protein isolated by the present inventors has a function of coloring a plant body or its seeds or accumulating tannins. The protein of the present invention is introduced into any plant species (such as rice varieties) and expressed. It is possible to color those plants or their seeds, or to accumulate tannins in the plants or their seeds. The period required for this transformation is extremely short compared to conventional gene transfer by crossing, and is advantageous in that it does not involve other phenotypic changes.

  As described above, the present invention also includes a method of coloring a plant or its seed, or accumulating tannins in the plant or its seed, including the step of expressing the DNA of the present invention in cells of the plant. It is. According to this method, a colored plant body or seeds with accumulated tannins are produced. Accordingly, the present invention provides a method for producing a colored or tannin-accumulated plant or a seed thereof, which comprises the step of expressing the DNA of the present invention in cells of the plant.

  More specifically, for example, white rice can be changed to colored rice such as red rice or reddish brown rice by the method of the present invention.

  The white rice varieties that can be colored by the method of the present invention are not particularly limited, and any white rice can be colored. Specifically, varieties such as Nipponbare, Koshihikari, Taichung No. 65, Akitakomachi and the like can be exemplified, but are not limited to these varieties.

  The present invention also provides a transformed cell into which the vector of the present invention has been introduced. The cells into which the vector of the present invention is introduced include plant cells for the production of transformed plants in addition to the cells used for the production of recombinant proteins. There is no restriction | limiting in particular as a plant cell, For example, cells, such as a rice, corn, wheat, and a barley, are mentioned. The plant cells of the present invention include cultured cells as well as cells in the plant body. Also included are protoplasts, shoot primordia, multi-buds, and hairy roots. For introduction of a vector into a plant cell, various methods known to those skilled in the art such as polyethylene glycol method, electroporation (electroporation), Agrobacterium-mediated method, and particle gun method can be used. Regeneration of plant bodies from transformed plant cells can be performed by methods known to those skilled in the art depending on the type of plant cells. For example, in rice, methods for producing transformed plants include a method for regenerating plants by introducing genes into protoplasts using polyethylene glycol, a method for regenerating plants by introducing genes into protoplasts using electric pulses, and a particle gun method. Some techniques have already been established, such as a method for directly introducing a gene into a cell and regenerating a plant, and a method for regenerating a plant by introducing a gene via Agrobacterium. Widely used. In the present invention, these methods can be suitably used.

  In order to efficiently select plant cells transformed by introduction of a vector containing the DNA of the present invention, the above recombinant vector contains an appropriate selection marker gene or is introduced into a plant cell together with a plasmid vector containing a selection marker gene. It is preferable to do. Selectable marker genes used for this purpose include, for example, the hygromycin phosphotransferase gene resistant to the antibiotic hygromycin, the neomycin phosphotransferase gene resistant to kanamycin or gentamicin, and the acetyl resistant to the herbicide phosphinothricin. Examples include transferase genes.

  Plant cells into which the recombinant vector has been introduced are placed on a known selection medium containing a suitable selection agent according to the type of the introduced selection marker gene and cultured. As a result, transformed plant cultured cells can be obtained.

  Transformed plant cells can regenerate plant bodies by redifferentiation. The method of redifferentiation varies depending on the type of plant cell. For example, for rice, the method of Fujimura et al. (Plant Tissue Culture Lett. 2:74 (1995)) can be mentioned, and for corn, Shillito et al. (Bio / Technology). 7: 581 (1989)) and Gorden-Kamm et al. (Plant Cell 2: 603 (1990)).

  Once a transformed plant into which the DNA of the present invention is introduced into the genome is obtained, offspring can be obtained from the plant by sexual reproduction or asexual reproduction. It is also possible to obtain a propagation material (for example, seeds, fruits, cuttings, tubers, tuberous roots, strains, callus, protoplasts, etc.) from the plant body, its descendants or clones, and mass-produce the plant body based on them. Is possible. The present invention includes a plant cell into which the DNA of the present invention has been introduced, a plant containing the cell, progeny and clones of the plant, and propagation material of the plant, its progeny and clones.

It is expected that the color of the plant thus produced or its seed is colored.
As described above, a method for producing a transformed plant comprising the steps of introducing the DNA or vector of the present invention into a plant cell and regenerating the plant from the plant cell is also included in the present invention.

  The colored plant of the present invention or its seed, or the plant or its seed in which tannins are accumulated can also be produced by a breeding method.

  Examples of the breeding method include a general breeding method (cross breeding method or the like) characterized by crossing with a variety having the DNA of the present invention. By this method, a colored plant body or its seed, or a plant body or its seed in which tannins are accumulated can be produced.

  When producing the plant body or seed of the present invention by the breeding method, it can be appropriately carried out with reference to various known documents. (Cellular engineering separate volume, Plant Cell Engineering Series 15 “Experimental Protocols for Model Plants” Shujunsha, 2001, Koji Kato “2.1 Mating of Rice and Wheat” p.6-9; Retsuro Takatsuki, Yoshio Sano “2 Mating method "p.46-48)

A preferred embodiment of the breeding method of the present invention is a method including the following steps (a) and (b).
(A) including the step of crossing with a plant having the DNA of the present invention,
(B) A step of selecting a plant variant having the DNA

More specifically, when the method of the present invention is carried out by a breeding method, a method including the following steps can be mentioned.
(A) crossing the plant A with another plant B having the DNA of the present invention to produce F1 (b) crossing the F1 with the plant A (c) selecting a plant having the DNA Step (d) A step of crossing the plant A selected by the step (c) with the plant A

  In the above method, the plant B having the DNA of the present invention is crossed with a plant to be colored or a plant to accumulate tannins (these plants are referred to as “plant A”), and the plant B has the present invention. An individual whose DNA is inherited and close to the plant A is selected, and a “backcross” in which the crossing by the plant A is repeated is intentionally introduced into the trait of the DNA of the present invention possessed by B. At that time, by using a DNA marker generally used for genome breeding to select a plant having the DNA of the present invention, it is possible to efficiently perform the replacement by the “backcross”. As a result, the breeding period can be shortened, and extra genomic region contamination can be accurately removed. Usually, in "backcrossing", there is a problem that a trait that depends on other DNA that is very strongly linked to the DNA of the present invention cannot be excluded, but the DNA present in the vicinity of the DNA of the present invention may be a problem. By using the marker, it is possible to accurately select a desired plant.

  The above method can be repeated as necessary until the entire genome other than the DNA of the present invention is homo-fixed with the desired genetic trait. That is, in a preferred embodiment of the present invention, the individuals crossed by the above step (d) have the DNA of the present invention using a general DNA marker and have a genome structure of plant A. Close plant individuals can be selected. Furthermore, the selected plant individual can be “backcrossed” (crossed with rice cultivar A) as necessary.

  In particular, in the genome breeding method using a DNA marker, individuals with a high replacement rate can be selected and proceed to the next cross, so that the selection efficiency increases as the generation progresses. In addition, in this method, since it is sufficient to handle a small number of individuals, it is possible to breed in a space-saving manner. Furthermore, it will be possible to cross multiple times a year using a greenhouse or climate chamber.

  In the above step (c), selecting using a DNA marker means selecting based on the base type information about the base sequence (for example, polymorphism) characterizing the DNA marker. For example, when a polymorphic mutation exists in the vicinity of the DNA of the present invention, it refers to selecting an individual having the same polymorphic mutation as the polymorphic mutation.

  The breeding method of the present invention is preferably a “genomic breeding” method using a DNA marker. The “genome breeding” is also called “marker breeding”.

  “Genome breeding” uses DNA markers that exist in high density throughout the entire genome, and depicts the situation of chromosome replacement for the entire genome of a progeny individual at each crossing. This is a breeding method in which an individual having sucrose is selected at the seedling stage and used for the next crossing. In conventional breeding methods, trait evaluation was used to select individuals to be used for the next cross, so it was necessary to grow to a large number of individual harvest periods, and crosses once a year or once every two years However, it took a great deal of labor and experience to cultivate a large number of individuals uniformly and to evaluate their characteristics correctly. Also, at the end of the breeding process, in order to make the entire chromosome homozygous, multiple self-breeding was necessary, and as a result, it took more than 10 years to complete the variety. This method relies on many years of experience and intuition. On the other hand, if the genome breeding strategy is used, germination of a large number of seeds obtained by crossing, selection of seedlings at the seedling stage, breeding a small number of individuals, and crossing are repeated. Since it is not necessary, it can be crossed several times a year using a greenhouse or a climate chamber. Furthermore, since whether or not the entire chromosome is homo-fixed can be confirmed in the selection process, the breed is usually completed when the crossing is completed. For this reason, it is possible to produce the target plant in a short period from the start. In addition, by sufficiently increasing the density of the DNA marker to be used, it is possible to introduce only the target gene region with an accuracy comparable to transformation. Since transformation is not used, it is possible to produce a plant that can be cultivated without problems in countries and regions where genetically modified crops are not accepted.

  As described above, a preferred embodiment of the breeding method of the present invention is a method characterized by selecting using a DNA marker present in the DNA sequence of the present invention or its peripheral sequence.

  The DNA marker that can be used in the breeding method of the present invention is not particularly limited, and various generally known DNA markers can be suitably used. For example, RFLP (restriction fragment length polymorphism) marker, SSR (simple repetitive sequence) marker, SNP (single nucleotide polymorphism) marker and the like can be exemplified.

  When the plant of the present invention is rice, for example, various markers shown in FIG. 21 can be used. Rc of the present invention is present between the two markers R1807-R1582, and is present in the vicinity of the marker of E12196S. Further, Rd (DFR) of the present invention exists between R2374 and C808. Therefore, preferred examples of the markers that can be used in the breeding method of the present invention include these four types of markers. It is of course possible to create markers in the DNA sequences identified this time.

  Further, by expressing the inventor's Rc or Rd gene in the plant, the plant body or its seed can be colored, and further, decolorization can be achieved by suppressing the expression of these genes in the colored plant body. It is also possible to produce (whitened) plants or their seeds.

  That is, the present invention is a gene endogenous to a plant, which encodes a protein having a function of coloring the plant body or its seed or a function of accumulating tannins in the plant body or its seed (the DNA of the present invention). A method for producing a decolorized plant body or a seed thereof, comprising a step of suppressing the expression of a gene comprising:

  The plant body is a genetically modified plant characterized in that the expression of the gene comprising the DNA of the present invention is artificially suppressed. The genetically modified plant is sometimes called, for example, a “knock-out plant”, “knock-down plant”, or “transgenic plant”.

  In the present invention, “gene expression is artificially suppressed” means, for example, (1) nucleotide insertion, deletion, substitution, etc. in one or both of the gene pairs in the gene comprising the DNA of the present invention. (2) the expression of the gene is suppressed by the action of a nucleic acid having a function of suppressing the expression of the gene of the present invention (for example, antisense RNA or siRNA). The state which is suppressed can be mentioned.

  In the “suppression” of the present invention, when the expression of the gene of the present invention is completely suppressed, the expression level of the gene of the present invention in the plant is significantly higher than the expression level of the gene in other plants. The case where it has fallen is included.

  The above (1) includes a case where only the expression of one gene of the gene pair of the present invention is suppressed. The site where the gene mutation is present in the present invention is not particularly limited as long as the expression of the gene is suppressed, and examples thereof include an exon site and a promoter site.

  The gene knockout plant of the present invention can be produced by those skilled in the art by generally known genetic engineering techniques. For example, a gene knockout plant can be produced as follows. First, DNA containing the exon portion of the gene of the present invention (Rc or Rd gene when the plant is rice) is isolated from the plant, and an appropriate marker gene is inserted into this DNA fragment to construct a targeting vector. This targeting vector is introduced into plant cells by the electroporation method, the Agrobacterium method, or the like, and a cell line in which homologous recombination has occurred is selected. The marker gene to be inserted is preferably a general drug selection gene. When a drug selection gene is inserted, cell lines that have undergone homologous recombination can be selected simply by culturing in a medium containing the drug. Alternatively, homologous recombinants can be assayed by PCR and Southern blotting to efficiently obtain a cell line in which the gene of the present invention is inactivated.

  The knockout plant (knockdown plant) of the present invention can also be produced, for example, by introducing a nucleic acid having a function of suppressing the expression of the gene of the present invention into the plant.

  Examples of the nucleic acid having a function of suppressing the expression of the gene of the present invention include the nucleic acids described in the following (a) to (c).

(A) an antisense nucleic acid for a transcription product of a gene comprising the DNA of the present invention or a part thereof (b) a nucleic acid having a ribozyme activity that specifically cleaves a transcription product of a gene comprising the DNA of the present invention (c) the present invention Nucleic acid having an inhibitory action by RNAi effect on the expression of gene consisting of

  The “nucleic acid” in the present invention means RNA or DNA. Further, chemically synthesized nucleic acid analogs such as so-called PNA (peptide nucleic acid) are also included in the nucleic acid of the present invention. PNA is obtained by replacing the pentose / phosphate skeleton, which is the basic skeleton structure of nucleic acids, with a polyamide skeleton having glycine as a unit, and has a three-dimensional structure very similar to nucleic acids.

  As a method for inhibiting the expression of a specific endogenous gene, a method using an antisense technique is well known to those skilled in the art. There are several factors as described below for the action of the antisense nucleic acid to inhibit the expression of the target gene. Inhibition of transcription initiation by triplex formation, inhibition of transcription by hybridization with a site where an open loop structure was locally created by RNA polymerase, inhibition of transcription by hybridization with RNA undergoing synthesis, intron and exon Splicing inhibition by hybridization at splice point, splicing inhibition by hybridization with spliceosome formation site, inhibition of translocation from nucleus to cytoplasm by hybridization with mRNA, hybridization with capping site and poly (A) addition site Inhibition of splicing by RNA, inhibition of translation initiation by hybridization with a translation initiation factor binding site, translation inhibition by hybridization with a ribosome binding site near the initiation codon, peptide by hybridization with an mRNA translation region or polysome binding site Elongation inhibition, and the like inhibiting gene expression by hybrid formation with the site of interaction between nucleic acids and proteins. In this way, antisense nucleic acids inhibit the expression of target genes by inhibiting various processes such as transcription, splicing or translation (Hirashima and Inoue, Shinsei Kagaku Kenkyu 2 Lecture and Expression of Nucleic Acid IV Gene, Japan Biochemical) (Academic Society, Tokyo Chemical Doujin, 1993, 319-347).

  The antisense nucleic acid used in the present invention may inhibit the expression of the gene of the present invention by any of the actions described above. As one embodiment, if an antisense sequence complementary to the untranslated region near the 5 ′ end of the mRNA of the gene of the present invention is designed, it is considered effective for inhibiting translation of the gene. In addition, a sequence complementary to the coding region or the 3 ′ untranslated region can also be used. Thus, the nucleic acid containing the antisense sequence of the sequence of the untranslated region as well as the translated region of the gene of the present invention is also included in the antisense nucleic acid used in the present invention. The antisense nucleic acid used is linked downstream of a suitable promoter, and preferably a sequence containing a transcription termination signal is linked on the 3 ′ side. The nucleic acid thus prepared can be transformed into a desired plant by using a known method. The sequence of the antisense nucleic acid is preferably a sequence complementary to the endogenous gene of the present invention or a part thereof possessed by the plant to be transformed. However, as long as the expression of the gene can be effectively suppressed, the sequence of the antisense nucleic acid is completely It does not have to be complementary. The transcribed RNA preferably has a complementarity of 90% or more, most preferably 95% or more, to the transcript of the target gene. In order to effectively suppress the expression of a target gene using an antisense nucleic acid, the length of the antisense nucleic acid is preferably at least 15 bases and less than 25 bases. However, the antisense nucleic acid of the present invention is not necessarily limited to this. It is not limited to length.

  In addition, the inhibition of the expression of the gene of the present invention can be carried out using a ribozyme or a DNA encoding the ribozyme. A ribozyme refers to an RNA molecule having catalytic activity. Some ribozymes have a variety of activities, but research focusing on ribozymes as enzymes that cleave RNA has made it possible to design ribozymes that cleave RNA in a site-specific manner.

  Hairpin ribozymes are also useful for the purposes of the present invention. It has been shown that hairpin ribozymes can also produce target-specific RNA-cleaving ribozymes (Kikuchi, Y. & Sasaki, N., Nucl Acids Res, 1991, 19, 6751., Hiroshi Kikuchi, Chemistry and Biology, 1992, 30, 112.). Thus, the expression of the gene can be inhibited by specifically cleaving the transcription product of the gene of the present invention using a ribozyme.

  Inhibition of endogenous gene expression can also be carried out by RNA interference (RNAi) using double-stranded RNA having the same or similar sequence as the target gene sequence. The nucleic acid having an inhibitory action by the RNAi effect of the present invention is generally also referred to as siRNA. RNAi induces destruction of target gene mRNA by introducing double-stranded RNA consisting of a sense RNA consisting of a sequence homologous to the mRNA of the target gene and an antisense RNA consisting of a complementary sequence into the cell. This is a phenomenon that can suppress the expression of the target gene. Since RNAi can suppress the expression of target genes in this way, it can be used as a simple gene knockout method to replace conventional complicated and low efficiency homologous recombination methods, or a method applicable to gene therapy. Has attracted attention as. The RNA used for RNAi is not necessarily completely identical to the gene of the present invention or a partial region of the gene, but preferably has complete homology.

  As a preferred embodiment of the nucleic acid (c) of the present invention, there can be mentioned double-stranded RNA (siRNA) having RNAi (RNA interference) effect on the gene of the present invention. More specifically, a double-stranded RNA (siRNA) composed of a sense RNA and an antisense RNA for the partial sequence of the base sequence described in SEQ ID NO: 2 or 5 can be mentioned.

  In addition, a molecule having one end closed in the above RNA molecule, for example, a siRNA (shRNA) having a hairpin structure is also included in the present invention. That is, a single-stranded RNA molecule capable of forming a double-stranded RNA structure within the molecule is also included in the present invention.

  The above-mentioned “double-stranded RNA that can be suppressed by the RNAi effect” of the present invention can be appropriately prepared by those skilled in the art based on the base sequence of the gene of the present invention that is the target of the double-stranded RNA. For example, the double-stranded RNA of the present invention can be prepared based on the nucleotide sequence shown in SEQ ID NO: 2 or 5. That is, based on the nucleotide sequence set forth in SEQ ID NO: 2 or 5, an arbitrary continuous RNA region of mRNA that is a transcription product of the sequence is selected, and a double-stranded RNA corresponding to this region is prepared. Those skilled in the art can appropriately carry out within the range of normal trials. In addition, selection of siRNA sequences having a stronger RNAi effect from mRNA sequences that are transcripts of the sequences can also be appropriately performed by those skilled in the art by known methods. Further, if one strand (for example, the base sequence described in SEQ ID NO: 2 or 5) is known, those skilled in the art can easily know the base sequence of the other strand (complementary strand). The siRNA can be appropriately prepared by those skilled in the art using a commercially available nucleic acid synthesizer. In addition, for synthesis of a desired RNA, a general synthesis contract service can be used.

  Furthermore, DNA (vector) capable of expressing the RNA of the present invention is also included in a preferred embodiment of the compound capable of suppressing the expression of the gene of the present invention. For example, the DNA (vector) capable of expressing the double-stranded RNA of the present invention is a DNA encoding one strand of the double-stranded RNA and a DNA encoding the other strand of the double-stranded RNA, Each DNA has a structure linked to a promoter so that it can be expressed. Those skilled in the art can appropriately prepare the above DNA of the present invention by general genetic engineering techniques. More specifically, the expression vector of the present invention can be prepared by appropriately inserting DNA encoding the RNA of the present invention into various known expression vectors.

  The knockdown plant of the present invention can be produced by introducing the nucleic acid (such as antisense RNA or siRNA) or a vector having a structure capable of expressing the nucleic acid into a desired plant.

  The present invention also provides a polynucleotide comprising at least 15 consecutive bases complementary to the base sequence set forth in SEQ ID NO: 1, 2, 4 or 5 or a complementary sequence thereof. Here, the “complementary sequence” refers to the sequence of the other strand with respect to the sequence of one strand of the double-stranded DNA comprising A: T and G: C base pairs. Further, the term “complementary” is not limited to the case where the sequence is completely complementary in at least 15 consecutive nucleotide regions, and is at least 70%, preferably at least 80%, more preferably 90%, and even more preferably 95%. What is necessary is just to have the identity of the above base sequence. Such DNA is useful as a probe for detecting the DNA of the present invention, selecting a plant body (plant cell) having the DNA of the present invention, and a primer for performing amplification of the DNA of the present invention. It is.

  The present invention also provides a reagent (for example, a plant breeding reagent) containing an oligonucleotide for use in the method for producing the plant of the present invention or its seed, or for the breeding method thereof.

More specifically, the reagent of the present invention is a reagent containing the following oligonucleotides (a) and (b).
(A) an oligonucleotide primer for amplifying the entire sequence of the DNA of the present invention or a partial sequence thereof (b) hybridizes with the DNA region of the present invention under stringent conditions and has a chain length of at least 15 nucleotides Oligonucleotide probe

  The primer or probe of the present invention can be synthesized by any method based on the base sequence constituting it. The length of the base sequence complementary to the genomic DNA of the primer or probe of the present invention is usually 15 to 100, generally 15 to 50, preferably 15 to 30. A technique for synthesizing an oligonucleotide having the base sequence based on the given base sequence is known. Furthermore, in the synthesis of the oligonucleotide, any modification can be introduced into the oligonucleotide using a nucleotide derivative modified with a fluorescent dye or biotin. Alternatively, a method of binding a fluorescent dye or the like to a synthesized oligonucleotide is also known.

  The present invention also provides a kit for use in the various methods of the present invention, for example, a breeding kit for use in the plant breeding method of the present invention.

  In a preferred embodiment of the kit of the present invention, the kit contains at least one kind of oligonucleotide (a) or (b). The kit of the present invention can be appropriately packaged with positive and negative standard samples, instructions describing the method of use, and the like.

  The DNA or vector of the present invention can be used, for example, for production of colored plants or colored seeds. Further, the DNA or vector of the present invention can be used for producing a plant body in which tannins are accumulated or seeds in which tannins are accumulated.

  By expressing the DNA or vector of the present invention in a desired plant or its seed, it is possible to produce a colored plant or its seed in which tannins are accumulated.

  Accordingly, the present invention provides a plant coloring agent comprising the DNA or vector of the present invention as an active ingredient.

  The “plant coloring agent” in the present invention refers to a drug having an action of coloring all or a part of a plant body or its seed, and is usually used for coloring a plant body or its seed. It refers to a substance or composition (mixture) containing the DNA or vector of the invention as an active ingredient.

  The present invention also provides a tannin accumulation agent comprising the DNA or vector of the present invention as an active ingredient.

  The “tannin accumulating agent” in the present invention refers to a drug having an action of accumulating tannins in all or a part of a plant body or its seed, and usually accumulates tannins in a plant body or its seed. Refers to a substance or composition (mixture) containing the DNA or vector of the present invention as an active ingredient.

  Furthermore, this invention provides the plant body decoloring agent which uses the nucleic acid as described in said (a)-(c) as an active ingredient.

  The “plant decoloring agent” in the present invention refers to a drug having an action of decolorizing all or a part of the plant or its seed, and is usually used for decolorizing the plant or its seed. It refers to a substance or composition (mixture) containing the nucleic acid of the present invention as an active ingredient.

  The “decolorization” refers to, for example, changing from a color such as red, reddish brown, or brown to white. When the plant is rice, it means that the color of rice, which is the seed, is changed from red rice to white rice, for example.

  In the drug of the present invention, in addition to DNA or vector as an active ingredient, for example, sterilized water, physiological saline, vegetable oil, surfactant, lipid, solubilizer, buffer, preservative, etc. are mixed as necessary. May be.

Moreover, this invention provides the plant body or its seed produced by the said manufacturing method or breeding method of this invention.
For example, (1) an artificially produced plant or seed thereof, which is characterized by having the DNA of the present invention and being colored, and (2) an artificially produced plant. Plant or seed thereof, wherein the plant body or seed thereof has the DNA of the present invention and tannins are accumulated, (3) a gene endogenous to the plant, Expression of a gene encoding a protein having a function of coloring the seed or a function of accumulating tannins in the plant or the seed is artificially suppressed and decolorized, or a plant thereof Seeds are included in the present invention.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples. The genotype symbols and scientific terms are the Kinoshita and Takahashi literature (Kinoshita, T. & Takahashi, M. (1991) J. Fac. Agr. Hokkaido Univ. 65, 1-61.) And the Kinoshita literature (Kinoshita , T. (1995) Rice Genetics Newsletter 12, 9-153.).

Example 1 Experimental Materials and Methods (a) Material Plant and Nucleic Acid Isolation Rice Nipponbare varieties, Kasaras, Koshihikari, tRc (NIL), purple rice, and transformed rice were cultivated in a 28 ° C. greenhouse. tRc is a near-isogenic line created by crossing CI-69 (red rice) with T-65 (chromosome 7 T-65 (genotype rcrd), chromosome 7 CI-69 (genotype RcRd ) Near-isogenic lines). Genomic DNA was extracted from young leaves 10 days after germination. For DNA extraction, DNeasy plant mini kit (Qiagen) was used and the protocol described in the kit was followed. Leaf RNA was extracted from young leaves 10 days after germination. Ear RNA was extracted from the ear on the 7th day after flowering. RNA extraction was performed using RNeasy plant mini kit (Qiagen) according to the method described in the kit.

(B) PCR, DNA sequence
The genomic DNA of OsDFR was amplified by 1U LA Taq DNA polymerase (TaKaRa). Primers used for PCR were OsDFRF4 (5′-CATGTGTGAGCATCTCTAAGTGACG-3 ′ / SEQ ID NO: 9) upstream of the translation initiation codon of the OsDFR gene and OsDFRR4 (5′-CTCTCTAGGAGCACGTGTAAAGG-) downstream of the translation termination codon of the DFR gene 0.5 kb. 3 ′ / SEQ ID NO: 10) was used (FIG. 4). The DFR PCR product was sequenced using the CEQ Dye Terminator Cycle Sequencing Start Kit and the CEQ 2000XL DNA Analysis System (Beqman Coulter) according to the method described therein. As a primer used for the sequence, OsDFRR5 (5′-AGAAGTCGATGTCGCTCCAG-3 ′ / SEQ ID NO: 11) corresponding to the second exon of the OsDFR gene was used (FIG. 4).

(C) RT-PCR
Synthesis of cDNA used as a substrate for PCR was performed using SuperScript ^™ reverse transcriptase and Random Primers (Invitrogen) and following the method described therein. PCR was carried out using Ampli taq (Applied Biosystems) at 94 ° C for 1 minute, and then a cycle of 94 ° C for 15 seconds, 60 ° C for 30 seconds, and 72 ° C for 30 seconds was repeated 40 times. The primers used were OsDFRF8 (5′-GATTCGCGTGCGAATCCAAC-3 ′ / SEQ ID NO: 12) corresponding to the first exon of the OsDFR gene, and OsDFRR5 (5′-AGAAGTCGATGTCGCTCCAG-3 ′ / corresponding to the second exon of the OsDFR gene. SEQ ID NO: 13) was used (FIG. 5). For amplification of rice actin gene (X15865) transcript, RAc1cDNA-F primer (5'-TGTCATGGTCGGAATGGG-3 '/ SEQ ID NO: 14) corresponding to the second exon of rice actin gene, and third exon of rice actin gene RAc1 cDNA-R primer (5′-TGCCAGGGAACATAGTGG-3 ′ / SEQ ID NO: 15) was used. PCR products were separated and identified by 1.8% (w / v) agarose gel electrophoresis.

(D) Protein Blot Analysis Total protein in the ear was extracted with extraction buffer (200 mM Tris-HCL.pH 6.8; 8% (w / v) SDS; 40% Glycerol; 5 mM 2-mercaptoethanol). The extracted sample was centrifuged at 4,000 × g for 5 minutes at 4 ° C., and the supernatant was dissolved in SDS-PSGE running Buffer. Proteins were separated on a 12% polyacrylamide gel and transferred to an Immobillon PVDF membrane.

  As an antiserum against DFR, a cDNA corresponding to the full length ORF of purple OsDFR was amplified by PCR, ligated to pGEX4T-3 vector (Amersham Pharmacia Biotech), and introduced into E. coli JM109 strain (FIG. 6). The GST / DFR fusion protein was induced by adding 1 mM IPTG (Asano, T., Kusano, H., Okuda, T., Kubo, N., Shimada, H. & Kadowaki, K. ( 2001) Purified according to the method described in Plant Cell Physial 43, 668-674.). The purified fusion protein was injected into rabbits 6 times every 2 weeks, and the serum was used for analysis.

(E) Rc genome annotation
Search for candidates using Rc genome annotation was performed using Rice GAAS (Rice Genome Automated Annotation System) {http://ricegaas.dna.affrc.go.jp/}.

(F) Production of transformed plants For the production of overexpressed murasakiine-type DFR and Koshihikari-type DFR, the genomic region corresponding to the full-length ORF of OsDFR of murasakiine and Koshihikari is in-frame downstream of the 35S promoter of pCAMBIA1301 plasmid. Were connected in the sense direction (FIGS. 7 and 8). For the production of DFR antisense individuals, the region corresponding to the ORF of OsDFR cDNA was ligated in the antisense direction downstream of the 35S promoter of pCAMBIA1301 plasmid (FIG. 9). Three candidates of Rc (Rc-MYB1, Rc-MYB2, and Rc-MYC) obtained by genome annotation are about 12 kb from the predicted promoter region of 1.5 kb to the predicted polyA addition signal of pCAMBIA1301 plasmid. Ligated to the multiple cloning site (FIG. 10). The genes used for the gene construction of Rc-MYB1, Rc-MYB2, and Rc-MYC were 105659-107804 (AP005197), 118440-1226019 (AP003754), and 150211-156639 (AP005779) derived from Casalas, respectively. The prepared plasmid was introduced into Agrobacterium tumefaciens strain EHA105. Transformation of rice by the Agrobacterium method (transformation of DFR was performed on tRc (NIL) and transformation of Rc was performed on Nipponbare) is described in Patent No. WO01 / 06844 Followed the method. The resulting transformed plant was grown in a greenhouse.

[Example 2] Identification of Rd gene (1) Phenotypes of RcRd, Rcrd, rcRd, and rcrd genotypes In rice, there are two control factors, Rc and Rd, for seed coat coloring by tannins (CTs). (Nagao, S. (1951) Advances in Genetics 4, 181-212.). Rc is an essential factor for the red and reddish brown coloring of the seed coat. Rd functions cooperatively with Rc only when Rc is present, and is an essential factor for red coloring of the seed coat. As a result of the hybridization experiment, it is known that the brown rice color of the RcRd genotype is red, the brown rice color of Rcrd is reddish brown (there is a hollow, and there is a deep coloring), rcRd and the brown rice color of rcrd is white (Fig. 3).

(2) Isolation of Rd locus with DFR
It is known from genetic analysis that the brown rice color of RcRd is red, the brown rice color of Rcrd is reddish brown, and the brown rice color of rcRd and rcrd is white (Nagao, S. & Takahashi, M. (1947) Jap. J. Genet. Supp. 1, 1-27.). In addition, the brown pigment of RcRd and Rcrd contains proanthocyanidins, which are the main pigments. Nagao et al. (Nagao, S., Takahashi, M. & Miyamoto, T. (1956) The Japanese Jourant of Genetics 32, 124-128 .) Confirmed. Since it affects the color of brown rice in rice, Rd is a locus involved in proanthocyanidin synthesis, and the allele rd does not lack a function. Compared with Rd, the proanthocyanidin synthesis system It is considered that the function in is weak. Here, from the report of Kinoshita et al. (Kinoshita, T. & Takahashi, M. (1991) J. Fac. Agr. Hokkaido Univ. 65, 1-61.), The Rd locus exists in the vicinity of the A locus, which is 0.3. Within cM (Figure 2).

  The A locus is a gene defined as an activator of rice anthocyanin synthesis system, and the gene encoded by the A locus has not been clarified so far. Coloring by rice anthocyanins has three genes, C (Chromogen), a chromogen gene, A (Activator), an anthocyanin activator, and P (Purple), which defines the coloring site. When these three factors are present, rice tissues (brown rice, leaves, lamina joints, stems, etc.) are colored by anthocyanins (Table 1).

( ^* Nagao et al. (Nagao, S. & Takahashi, M. (1963) Agr., Hokkaido Univ., Sapporo 53, 76-131.) And Kato et al. (Kato, S., & Ishikawa. J. (1923) Jap The locus determined by J. Genet. 1, 1-7.) Is an independent study and cannot be compared.)

  Here C is known to be a homologue of corn C1 (MYB) (Reddy, VS, Scheffler, BE, Wienand, U., Wessler, SR & Reddy, AR (1998) Plant Molecular Biology 36, 497 Saitoh, K., Onishi, K., Mikami, I., Thidar, K. & Sano, Y. (2004) Genetics 168, 997-1007.), P is corn R / B (bHLH) (Hu, J., Anderson, B. & Wessler, RS (1996) Genetics 142, 1021-1031 .; Hu, J., Reddy, SV & Wessler, RS (2000 ) Plant Molecular Biology 42, 667-678 .; Sakamoto, W., Ohmori., T., Kageyama, K., Miyazaki, C., Saito, A., Murata, M., Noda, K. & Maekawa, M (2001) Plant Cell Physiol. 42, 982-991.) (Table 2).

  This indicates that the rice A gene encodes DFR.

  Rd exists in the vicinity of A (DFR) (Nagao, S. & Takahashi, M. (1947) Jap. J. Genet. Supp. 1, 1-27.). As a result of analyzing the gene present between R2374 (103.7 cM) and C808 (106.2 cM) where A (DFR) exists using the Rice GAAS program, there is no gene involved in tannin synthesis other than DFR in this region. There wasn't. Therefore, it was suggested that Rd also encodes DFR like A.

  From the above results, it was hypothesized that the Rd gene and the A gene were the same, and these genes encoded the same DFR protein. In order to test this hypothesis, a mating experiment was performed between the near-isogenic line T-65 (RcRd, A) and the tRc line (Rcrd, a). If the A and Rd genes are not identical, a recombinant that occurs between A and Rd is observed in F2. The recombinant has a genotype of (Rcrd, A) or (RcRd, A).

  Since the A gene is a causative gene for the anthocyanin pigment in the tip (apiculus), the plant having (Rcrd, A) has a tip that has a red-brown seed skin and is colored by anthocyanin. On the other hand, a plant having (RcRd, a) has a red skin of the seed and has a non-colored tip.

  As a result of the experiment, in F2, 672 individuals had a red seed skin, and a tip colored with anthocyanins by (RcRd, A) was observed. In 232 individuals, the skin of the seeds became reddish brown, and a non-colored tip due to (Rcrd, a) was observed. No recombinant between Rd and A was observed. This suggests that Rd and A are strongly genetically linked and are the same gene.

(3) Complementarity of Rcrd and genomic OsDFR
As a result of complementing tRc (Rcrd) with DFR derived from purple rice, the brown rice color changed from reddish brown to red. Considering that the brown rice color of RcRd is red, it became clear that Rd encodes DFR (FIG. 11).

(4) Analysis of DFR allele
In order to investigate the relationship between the seed coat color depending on the genotype of Rd and rd and the structure of the DFR gene, DNA sequencing was performed. The analysis uses Kasalath with the RcRd genotype, tRc with the Rcrd genotype, and Nipponbare and Koshihikari with the rcrd genotype. The region from the first exon to the second exon of DFR was amplified by PCR using genomic DNA as a template and subjected to direct sequencing. As a result, Kasalath with Rd had a structure similar to the previously isolated rice DFR gene (Chen, M. & LJ Bennetzen (1996) Plant Molecular Biology 32, 999-1001 .; Nakai, K., Inagaki, Y., Nagata, H., Miyazaki, C., & Iida, S. (1998) Plant Biotechnology 15, 221-225.). On the other hand, in the structure of tRc with rd and DFR gene of Nipponbare, there was a translation stop codon (TAG) that seems to have been created by mutation in the second exon. Furthermore, the Koshihikari DFR structure, which was thought to have rd, had a translation termination codon (TAG) that was thought to have been formed by mutation immediately downstream of the start codon of the first exon, unlike Kasalas and Nihonbare (Fig. 12). ). From the above, it has been clarified that Rd encodes Kasaras type DFR, and rd encodes Koshihikari and Nipponbare type DFRs in which translation stop codons exist in the first and second exons.

  The structure of rd's DFR gene is that there is a stop codon in the second exon, and from the start codon to the translation stop codon made by mutation is 4.5 kDa, whereas OsDFR encodes 42 kDa. It is hard to think of having. There is only one copy of DFR in rice (Nakai, K., Inagaki, Y., Nagata, H., Miyazaki, C., & Iida, S. (1998) Plant Biotechnology 15, 221-225 .; International Rice Genome Sequence Project. (2005) Nature 436, 793-800.). However, the DFR gene of Rcrd is Nipponbare type, and the N-terminal side of ORF is deficient, but the brown rice color is reddish brown and is known to be colored by proanthocyanidins (Nagao, S., Takahashi, M. & Miyamoto, T. (1956) The Japanese Jourant of Genetics 32, 124-128.). Therefore, it was considered that the Nipponbare type DFR having a stop codon in the second exon has a function. Therefore, in order to examine whether DFR, an Rd allele, is transcribed, RT-PCR was performed on Kasalath with the RcRd genotype, Nipponbare with the rcrd genotype, and Koshihikari with the rcrd 'genotype. went. As a result, Kasalath, tRc, Nipponbare, and Koshihikari were specifically expressed in the ears on the 7th day after flowering, and the strength of the expression level was Kasaras> Nipponbare> Koshihikari (FIG. 13).

  Next, in order to investigate the mechanism of translation of DFR, which is the Rd allele, the present inventors have investigated Kasalath with the RcRd genotype, Nipponbare with the rcrd genotype, and Koshihikari with the rcrd genotype. Then, immunostaining was performed on the 7th day after flowering. Surprisingly, Kasaras was found to have an alternative translation initiation (Bab, I., Smith, E., Gavish, H., Malka, Attar-Namdar., Chorev, M., Yu-Chen, Chen., Muhlrad, A ., Birnbaum, JM, Stein, G. & Frenkel, B. (1999) The Journal OF BIOLOGICAL CHEMISTRY 274, 14474-14481 .; Chabregas, MS, Luche, DD, Marie-Anne, Van, Sluys., Carlos, FM Menck., Marcio, C. Silva-Filho. (2002) Journal of Cell Science 116, 285-291 .; Smith, E., Meyerrose, ET, Kohler, T., Malka, Namdar-Attar., Bab, N. , Lahat, O., Noh, T., Li, J., Karaman, WM, Hacia, GJ, Chen, TT, Nolta, AJ, Muller, R., Bab, I. & Frenkel, B. (2005) Nucleic Acids Research 33, 1298-1308 .; Hanfrey, C., Elliott, AK, Franceschetti, M., Mayer, JM, Illingworth, C. & Michael, JA (2005) The Journal OF BIOLOGICAL CHEMISTRY, in press .; Kabeya, Y. & Sato, N. (2005) Plant Physiology 138, 369-382.), There were three ORFs of 46 kDa, 33 kDa and 22 kDa. In addition, there were two ORFs of 33 kDa and 22 kDa in Nipponbare and Koshihikari. This 46 kDa band was larger than the expected protein size, but it was thought that the band was shifted because there were many basic amino acids on the N-terminal side of DFR (FIG. 4).

  Here, there are 5 Kozak consensus sequences in the ORF of OsDFR including the first start codon (Kozak, M. (2000) Genomics 70, 396-406 .; Kozak, M. (2002) Gene 299, 1 -34.). The 46 kDa and 22 kDa bands are detected relatively strongly, but this is the first and fourth with GnnATGG (n is any base of A, T, G, or C) with the strongest Kozak consensus sequence. It was considered a Kozak consensus. Regarding the origin of the 33 kDa band, it was speculated that translation was initiated from ATG other than the Kozak sequence of the second exon, considering the size of the molecular weight (FIG. 4).

  Based on the above results, rd type DFR was translated from downstream ATG by Alternative translation initiation (Kozak, M. (2000) Genomics 70, 396-406 .; Kozak, M. (2002) Gene 299, 1-34.) And was shown to complement the function of DFR. These results suggest that coloring with tannins occurs when Rc is complemented to Nipponbare with the rcrd genotype.

[Example 3] Identification of Rc gene (1) Genomic annotation of Rc By genetic analysis, Rc is known to exist on chromosome 7 (Nagao, S. & Takahashi, M. ( 1963) Agr., Hokkaido Univ., Sapporo 53, 76-131.) (FIG. 15A). The completion of the rice genome project revealed that Rc exists between two molecular markers, R1807 (42.6 cM) and R1582 (47.7 cM), on chromosome 7 (IRGSP, 2005) (FIG. 5B). Enzymes of the proanthocyanidin synthesis system (FIG. 1) existing in this region were searched using RiceGAAS (http://ricegaas.dna.affrc.go.jp/). However, no high homology exists for the enzyme of the proanthocyanidin synthesis system (FIG. 1) in this region.

  Next, we focused on the transcription factor of proanthocyanidin synthesis system. As transcription regulators of the proanthocyanidin synthesis system, Arabidopsis TT8 (Transparent Testa 8) (Nesi, N., Debeaujon, I., Jond, C., Pelletier, G., Caboche, M. & Lepiniec, L. (2001) Plant Cell 12, 1863-1878.), Arabidopsis TT2 encoding MYB related protein (Nesi, N., Jond, C., Debeaujon, I., Caboche, M. & Lepiniec, L. (2001) Plant Cell 13, 2099-2114.) Is known. Therefore, the genes encoding MYC type protein and MYB related protein present in R1807 (42.6 cM) and R1582 (47.7 cM) on chromosome 7 where Rc is present were searched using RiceGAAS.

  As a result, two MYB related proteins and one MYC type protein were found. One of the genes encoding MYB related protein was named Rc-MYB1, and ORG was present at 105659 bp-107804 bp of AP005197 of IRGSP. Another MYB related protein was named Rc-MYB2, and ORF was present at 118440 bp-126019 bp of AP003754 of IRGSP. The MYC type protein was named Rc-MYC, and its ORF was present at 150211 bp-156639 bp of AP005779 of IRGSP (FIGS. 15A, B, C). Rc-MYB1 and Rc-MYB2 both had a homology of 24% (Identities) with TT2 (MYB related protein), a transcriptional regulator of the Arabidopsis proanthocyanidin synthesis system. On the other hand, Rc-MYC has a homology of 38% (Identities) with TT8 (MYC type protein), a transcription factor of Arabidopsis proanthocyanidin synthesis system, and a part of the bHLH domain has a high homology of 73% ( Identities) (FIG. 15D) (ClustalW ver.1.82).

(2) Complementarity of rcrd and genome fragment
Rc-MYB1, Rc-MYB2, and Rc-MYC of Casalas (RcRd) with the RcRd genotype (Fig. 15) rcrd about 12 kb from the translation initiation codon 1.5 kb upstream of the promoter region to the poly A addition signal. The gene was introduced into Nipponbare (rcrd) with genotype. 16 individual transformants were obtained. For the individuals into which Rc-MYB1 and Rc-MYB2 had been introduced, coloring of proanthocyanidins was not observed in leaves, fir or brown rice. On the other hand, in the individuals into which Rc-MYC was introduced, the brown rice was colored to be considered to be due to proanthocyanidins (FIG. 16). Ten transformants with coloration were obtained, and their phenotype was very similar to tRc with the Rcrd genotype.

  From the above results, it was shown that Rc-MYC is Rc, which is a proanthocyanidin synthesis system controlling factor. Coloration was not seen in tissues other than brown rice. From the above, it was shown that Rc is a transcription factor that works specifically for seeds.

(3) Analysis of Rc region Next, the present inventors determined the full length cDNA sequence of Rc-MYC of Kasalath (RcRd). The RNA used was extracted from the ear on the 7th day after flowering. As a result of sequencing the full-length Kasalath Rc-MYC, it was found that the Kasalath Rc-MYC is a gene consisting of 8 exons encoding a protein of 673 aa.

  Next, the present inventors decided to compare the DNA sequence of Rc-MYC of Kasalath (RcRd) with the gene sequence of Nipponbare (rcrd). As a result, Nipponbare with rc was confirmed to have a 14 bp deletion mutation in the seventh exon, and a 6 bp insertion mutation and a 9 bp deletion mutation in the eighth exon. Nipponbare's 14-bp deletion mutation in exon 7 caused a frameshift, resulting in a stop codon 9 bp downstream of the 7th exon deletion site. As a result, Nipponbare Rc-MYC encoded a protein of 478 a.a. which was 215 a.a. shorter than Kasaras Rc-MYC. The bHLH domain required for DNA binding is encoded by the 8th exon. Therefore, Nipponbare's Rc-MYC has no bHLH domain because it is not translated due to a deletion mutation until the 8th exon. Therefore, it was considered that Nipponbare Rc-MYC cannot function as a transcriptional regulator (FIG. 18).

  Rc-MYC exon 7 14 bp deletion region, polymorphism analysis using sequence RcRd (brown rice color is red), Rcrd (brown rice color is red brown), rcrd (brown rice color is white) Went about. FIG. 19 shows that the brown rice color RcRd and the brown rice color Rcrd have no deletion in the seventh exon, and the brown rice color rcrd has a deletion in the seventh exon. The result of FIG. 19 suggests that coloring of brown rice with proanthocyanidins is due to Rc-MYC.

  Genetic analysis and reverse genetic analysis clearly showed that the brown rice color of Nipponbare became white due to the lack of Rc-MYC function.

(4) Homology analysis of MYC type protein Next, we decided to investigate the genetic distance between Rc-MYC and its homologue. The alignment of amino acid sequences was ClastalX ver1.83, and the phylogenetic tree was NJ method on clastalX. Boots strap value also used the function on ClastalX. Maize IN1 (U57899), Lc (M26227), B-Peru (X57276), RS (X15806), Petunia JAF13 (AF020545), Snapdragon DELILA (M84913), Arabidopsis TT8 ( AJ277509), rice Ra (U39860), Rc-MYC (this study). Phylogenetic analysis revealed that rice Rc-MYC had the highest homology with Intensifier (IN1), which was isolated as a maize anthocyanin synthesis inhibitor. However, although Rc-MYC is a tannin accumulation promoting factor, IN1 has the opposite function because tannin accumulates when its gene function is lost. On the other hand, it had the lowest homology with snapdragon DELILA and petunia JAF13 isolated as activators of anthocyanin synthesis system (FIG. 20).

It is a figure which shows the relationship between DFR and tannin synthesis and anthocyanin synthesis. Abbreviations of enzymes are as follows. F3H, flavanone 3-hydroxylase; DFR, dihydroflavonol-4-reductase; ANS, anthocyanin synthase; LAR, leucoananthocyanidin reductase; ANR, anthocyanidin reductase; BAN, BANYULS; GT, anthocyanidin glucosyl transferase It is a figure which shows the position of the gene in connection with the brown rice color of rice. The horizontal line represents the position of the marker. (A) Information on the initial seating position regarding Rc and Rd (Nagao, S. & Takahashi, M. (1963) Agr., Hokkaido Univ., Sapporo 53, 76-131.). As genetic markers on the first chromosome, A indicates an anthocyanin activator, and Pn indicates a purple node. As genetic markers on chromosome 7, d ₆ shows lop-leaved dwarf, g is a recessive long empty glumes respectively. (B) Rc, Rd sitting position information after the completion of the rice genome project (IRGSP 2005 Nature) R1807, R1582, R2374, C808 (http://rgp.dna.affrc.go.jp/publicdata/geneticmap2000/) Is a molecular marker described in. (C) Positions of Rc and Rd according to the present study (present invention). The Rd locus encodes the DFR gene, and its position on the chromosome is 68929 bp-70543 bp of AP004317 of the first chromosome. The Rc locus encodes a MYC related protein, and the position on the chromosome is AP005098 150211 bp-156639 bp of the seventh chromosome. It is a photograph which shows the genotype and phenotype of RcRd. The upper part of the photo is brown rice and the lower part is rice bran. (A) Brown rice color of Kasalath with RcRd genotype. (B) Brown rice color of tRc (NIL) with Rcrd genotype. (C) Nipponbare's brown rice color with rcrd genotype. The brown rice color with rcRd genotype is also white. (Nagao, S. (1951) Advances in Genetics 4, 181-212.) It is a figure which shows the structure of a rice DFR gene. Horizontal black squares indicate DFR exons. The horizontal lines between the black squares indicate the first intron and the second intron, respectively. OsDFRF4 and OsDFRR4 were used for PCR of the rice DFR gene. OsDFRR5 was used to determine the base sequences of the first exon, the first intron, and the second exon of rice DFR. It is a figure which shows the structure of rice DFR mRNA. The black line shows the ORF. OsDFRF8 and OsDFRR5 were used for RT-PCR of rice DFR. It is a figure which shows DFR (pGEX4T-3 DFR) connected with the pGEX4T-3 vector. It was constructed based on pGEX4T-3. AmpR represents an ampicillin resistance gene, Ptac represents a tac promoter, and GST represents glutathione-S-transferase. BamHI and EcoRI are restriction enzymes used in preparing the vector. FIG. 3 shows the structure of a 35S :: Murasaki DFR vector. It was constructed based on pCAMBIA1301 (vector part: 11.8 kb). LB and RB indicate a left border and a right border, respectively. T35S and P35S are promoters for strong expression derived from cauliflower mosaic virus. MCS indicates a multiple cloning site. NosT represents a terminator derived from nopaline synthase. HygR and KanR are resistance genes for hygromycin and kanamycin, respectively. Murasakiine DFR is the genome sequence (including intron) of DFR of purple rice. FIG. 3 shows the structure of a 35S :: Koshi DFR vector. It was constructed based on pCAMBIA1301 (vector part: 11.8 kb). LB and RB indicate a left border and a right border, respectively. T35S and P35S are promoters for strong expression derived from cauliflower mosaic virus. MCS indicates a multiple cloning site. NosT represents a terminator derived from nopaline synthase. HygR and KanR are resistance genes for hygromycin and kanamycin, respectively. Koshihikari DFR is the genome sequence (including introns) of Koshihikari DFR. It is a figure which shows the structure of AsDFR vector. It was constructed based on pCAMBIA1301 (vector part: 11.8 kb). LB and RB indicate a left border and a right border, respectively. T35S and P35S are promoters for strong expression derived from cauliflower mosaic virus. MCS indicates a multiple cloning site. NosT represents a terminator derived from nopaline synthase. HygR and KanR are resistance genes for hygromycin and kanamycin, respectively. Murasakiine DFR is the purple rice DFR cDNA sequence (without intron). It is a vector that suppresses the expression of DFR by Antisense method. FIG. 3 is a diagram showing the structures of pRc1-1301, pRc2-1301, and pRc3-1301 vectors. It was constructed based on pCAMBIA1301 (vector part: 11.8 kb). LB and RB indicate a left border and a right border, respectively. T35S and P35S are promoters for strong expression derived from cauliflower mosaic virus. MCS indicates a multiple cloning site. NosT represents a terminator derived from nopaline synthase. HygR and KanR are resistance genes for hygromycin and kanamycin, respectively. Gus gene indicates the Gus gene. promoter indicates a promoter derived from Rc1, Rc2, and Rc3, respectively. T represents a terminator derived from Rc1, Rc2, and Rc3, respectively. It is a photograph showing the phenotype of an individual into which DFR has been introduced. (A) Phenotype of an individual transgenic with 35S :: Murasaki DFR. The top represents a photograph of immunostaining using DFR antibody on the 7th day after germination. The second row represents the 7th day after heading. The third tier represents fully ripe seeds. (B) The phenotype of the individual into which As DFR was introduced. The top represents a photograph of immunostaining using DFR antibody on the 7th day after germination. The second row represents the 7th day after heading. The third tier represents fully ripe seeds. (C) tRc (Rcrd) phenotype. The second row represents the 7th day after heading. The third tier represents fully ripe seeds. It is a figure which shows the polymorphism of a DFR gene. Black squares indicate exons. Horizontal lines indicate introns. Open circles indicate translation initiation codons. Open squares indicate translation stop codons. It is a photograph which shows RT-PCR of a rice DFR. DFR PCR was performed for 35 cycles. Actin PCR was performed for 30 cycles. The number on the right is the size of the amplified fragment. It is the figure and photograph which show the comparison of the expression of DFR. (A) Nipponbare, Kasaras DFR structure. A horizontal black square represents an exon. The horizontal black line represents the intron. Open circles represent start codons with Kozak consensus (ANNATGN or GNNATGG). The upward triangle indicates the position of the intron. The open square represents the position of the translation stop codon. (B) ORF (Alternative translation initiation) predicted from the structure of OsDFR. The horizontal line shows the expected ORF and the number on the right represents the expected protein molecular weight. (C) and (D) are photographs showing immunostaining of DFR in ears at the ripening stage and leaves on the seventh day after germination. (C) GST-DFR (positive control); Kasaras; Nipponbare; Koshihikari extracted total protein and immunostained with Anti-DFR antibody. The numbers on the right indicate the molecular weight of the protein. (D): GST-DFR (positive control); 35S :: DFR transgenic animal; Kasaras; Nipponbare; Koshihikari extracted all proteins and immunostained with Anti-DFR antibody. The numbers on the right indicate the molecular weight of the protein. It is a figure which shows the annotation of Rc using the RiceGAAS program. (A) Shows the position of Rc on chromosome 7. (B) MYC type protein and MYB related protein that exist between R1807 and R1582 where Rc is considered to be seated. White circles indicate molecular markers. Arrows indicate MYC type protein (predicted gene) and MYB related protein (predicted gene), respectively. (C) Precise mapping of Rc. White circles indicate molecular markers. The bottom row shows the Nipponbare clone of the Rice Genome research program. The arrow indicates the position of the Rc gene predicted from the database search. The black square is the region used for the Rc complementation experiment. Rc (invention) and TT8 (Nesi, N., Debeaujon, I., Jond, C., Pelletier, G., Caboche, M. & Lepiniec, L. (2001) Plant Cell 12, 1863-1878.) It is a figure which shows alignment of a predicted amino acid sequence. Identical amino acids are indicated by black squares and similar amino acids are indicated by boxes. The horizontal black line shows the bHLH domain unique to MYC type protein. It is a photograph which shows the complementary experiment result of Rc. The upper part of the photo is brown rice and the lower part is rice bran. (A) Kasalath with RcRd genotype. (B) Non-transformant. (C) Nipponbare with rcrd genotype. (D) Rc-MYC-16 (NO.16 of a plant complemented with Rc-MYC in Nipponbare with the rcrd genotype). (E) Rc-MYC-23 (NO.23 of a plant that complemented Rc-MYC with Nipponbare with the rcrd genotype). (F) Rc-MYC-13 (NO.13 of a plant with Rc-MYC complemented to Nipponbare with the rcrd genotype). (G) tRc with Rcrd genotype. (H) Rc-MYB1 (a plant with Rc-MYB1 complemented to Nipponbare with the rcrd genotype). (I) Rc-MYB2 (a plant in which Rc-MYB2 is complemented to Nipponbare with the rcrd genotype). It is a figure which shows the control model of the brown rice color by Rc and Rd. It is a figure which shows the structure (casalas) of Rc-MYC. Boxes indicate exons and horizontal lines indicate introns. An upward black triangle indicates a deletion in Nihonbare, and a downward white triangle indicates an insertion mutation in Nihonbare. It is a figure which shows the polymorphism analysis of the deletion mutation of 14 bp which exists in the 7th exon of Rc-MYC. Samples used were O. sativa with brown RcRd genotype, brown rice color, Indica type Kasalath, Japonica type Toboshi (TOBOSHI), Akamai Asahi (AKAMAIASAH), Akamai (AKAMAI), Homan Nitta DAMA / THAI / 86/148 (ORD) and COL / THAILAND / 1990 / MAFF / 181 (ORC) were used from rice (HOUMAN) and Danema (DANEMA) O.rufipogon. For the brown rice with a reddish brown Rcrd genotype, NIL tRc and COL / KAGOSHIMA / 1963 MAJIRI / 1187 (col-kago) were used. For those with brown rice rcrd genotypes, Nihonbare (nipponbare), Koshihikari (Koshi), Taichung 65 (T65), Dougowase (DOUGO), Mitonishiki (MITONISHIKI), FUSAKUMOCHI, Shimizu Shiraz (SHIMIZU) Was used. Since the relationship between coloring (red and reddish brown) and white coincides with the presence or absence of the deletion, the involvement of the gene in coloring was proved in addition to the results of FIG. It is a figure which shows the systematic analysis of Rc-MYC. Amino acid sequences with bHLH domains were aligned using Clastal X. The creation of the phylogenetic tree and the Boots strap value were calculated using the NJ method on Clastal X and the most saving method. IN1, Lc, R-S, and B-Peru are corn, Ra is rice, TT8 is Arabidopsis, JAF13 is petunia, and DELILA is an anthocyanin synthesis regulator of goldfish. FIG. 3 is a view showing a specific example of a DNA marker that is present in the vicinity of the Rc (MYC) and Rd (DFR) genes of the present invention and can be used in the breeding method of the present invention.

Claims (30)

The DNA according to any one of the following (a) to (e), which encodes a plant-derived protein having a function of coloring a plant body or its seed or a function of accumulating tannins in the plant body or its seed.
(A) DNA encoding a protein comprising the amino acid sequence of SEQ ID NO: 3 or 6-8.
(B) DNA comprising the coding region of the nucleotide sequence set forth in SEQ ID NO: 1 or 4.
(C) DNA comprising the nucleotide sequence set forth in SEQ ID NO: 2 or 5
(D) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence of SEQ ID NO: 3 or 6-8.
(E) A DNA that hybridizes under stringent conditions with a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1 or 4.   The DNA according to claim 1, wherein the plant is a monocotyledonous plant.   The DNA according to claim 2, wherein the monocotyledonous plant is rice.   A protein encoded by the DNA according to any one of claims 1 to 3.   A vector comprising the DNA according to any one of claims 1 to 3.   A host cell into which the vector according to claim 5 has been introduced.   A plant cell into which the vector according to claim 5 has been introduced.   A transformed plant comprising the plant cell according to claim 7.   A transformed plant that is a descendant or clone of the transformed plant according to claim 8.   The propagation material of the transformed plant body of Claim 8 or 9.   The method for producing a protein according to claim 4, comprising a step of culturing the host cell according to claim 6 and recovering the recombinant protein from the cell or a culture supernatant thereof.   An antibody that binds to the protein of claim 4.   A polynucleotide comprising at least 15 consecutive bases complementary to the base sequence of the DNA according to any one of claims 1 to 3 or a complementary sequence thereof. The plant body coloring agent which contains either of following (a)-(c) as an active ingredient.
(A) DNA according to any one of claims 1 to 3
(B) The protein according to claim 4 (c) The vector according to claim 5 A tannin accumulating agent containing any of the following (a) to (c) as an active ingredient.
(A) DNA according to any one of claims 1 to 3
(B) The protein according to claim 4 (c) The vector according to claim 5 The plant body decoloring agent which contains either of following (a)-(c) as an active ingredient.
(A) an antisense nucleic acid for the transcription product of the DNA according to any one of claims 1 to 3 or a part thereof; (b) specifically cleaving the transcription product of the DNA according to any one of claims 1 to 3; Nucleic acid having ribozyme activity (c) Nucleic acid having an inhibitory action due to RNAi effect on expression of DNA according to any one of claims 1   A method for producing a transformed plant comprising the steps of introducing the DNA according to any one of claims 1 to 3 or the vector according to claim 5 into a plant cell and regenerating the plant from the plant cell.   A method for coloring a plant or its seed, or accumulating tannins in the plant or its seed, comprising the step of expressing the DNA according to any one of claims 1 to 3 in a cell of the plant.   A method for producing a colored or tannin-accumulated plant or a seed thereof, comprising the step of expressing the DNA according to any one of claims 1 to 3 in a cell of the plant.   The method of Claim 18 or 19 including the process of introduce | transducing the DNA in any one of Claims 1-3, or the vector of Claim 5 into a plant cell. The method according to claim 18 or 19, comprising the following steps (a) and (b).
(A) including a step of crossing with a plant having the DNA according to any one of claims 1 to 3,
(B) A step of selecting a plant variant having the DNA A method for breeding a plant having a colored seed or a seed having accumulated tannins, the method comprising the following steps (a) to (d):
(A) crossing the plant A and another plant B having the DNA according to any one of claims 1 to 3 to produce F1 (b) crossing the F1 and the plant A (c) A step of selecting a plant having the DNA (d) a step of crossing the plant A selected by the step (c) with the plant A   In the step (c), the DNA sequence according to any one of claims 1 to 3 in the plant genome or a DNA marker existing in the peripheral sequence is used for selection. The method described in 1.   Decolorization comprising the step of suppressing the expression of a gene that is endogenous to a plant and that encodes a protein having a function of coloring the plant body or its seed or a function of accumulating tannins in the plant body or its seed For producing a plant body or a seed thereof.   The method according to any one of claims 17 to 24, wherein the plant is rice.   A plant obtained by the method according to any one of claims 18 to 25, or a seed thereof.   An artificially produced plant or seed thereof, wherein the plant or seed is characterized by having the DNA of any one of claims 1 to 3 and being colored.   An artificially produced plant or seed thereof, wherein the plant or seed thereof has the DNA of any one of claims 1 to 3 and tannins are accumulated therein.   Expression of a gene encoding a protein that is endogenous to a plant and has a function of coloring the plant body or its seed or a function of accumulating tannins in the plant body or its seed is artificially suppressed and decolorized A plant or its seed characterized by being made.   30. The plant body or the seed thereof according to any one of claims 27 to 29, wherein the plant is rice.