JP2011101622A - Gene increasing fruit of plant body and utilization thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide isolation and identification of a new gene associated with elongation of fruit grain (including a seed, a glumous flower or a fruit) of a plant body and, consequently the grain length, and to provide a breeding method for increasing the fruit of the plant body by utilizing the gene. <P>SOLUTION: A success is made in the isolation and identification of the GL5 gene associated with the elongation of the fruit (including the seed, glumous flower or fruit) of the plant body and, consequently an increase in yield by a linkage analysis. Furthermore, it is revealed that the fruit of a monocotyledon or a dicotyledon can be increased by utilizing the gene. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a DNA encoding a protein derived from rice having a function of increasing the grain (including seeds, spikelets or fruits) of a plant body, and the grain yield (seed, spikelets or fruits) using the DNA. And a method for detecting grain yield, and a method for breeding a plant that increases grain yield.

  The world population has exploded and there is a demand for increased production of crops (for example, grains). The global population growth rate is 1.4%, while the crop production growth rate is 1.0%, which is lower than the population growth rate. By 2025, when the world population exceeds 8 billion, crop demand is expected to rise by 50%. The food shortage is expected to accelerate further. In order to increase crop production, it is essential to identify the genes involved in yield increase and efficient grain breeding using the identified genes.

  The grain size of rice is a value indicating the capacity of the sink and directly affects the yield. So far, GW2 (Non-patent document 1) and GS3 (Non-patent document 2) have been isolated by the positional cloning method as genes related to seed (grain) size in rice. However, these genes do not contribute to increased yield. In addition, transgenic plants in which the plastchron 1 gene is expressed and the grains are enlarged, and a method for using the transgenic plants (Patent Document 1), and a method in which the modified trimer G protein α subunit gene is used to produce a large grain (Patent Document 2). Patent application has been filed for the Lk3 gene that controls the grain length of rice and its use (Patent Document 3). Furthermore, the present inventors have isolated a gene (TGW6) that increases the grain weight and thus the yield (Patent Document 4).

However, genes isolated to date have not been sufficient to increase rice seeds. Also, it was not clear how to increase grain beyond species.
Prior art document information related to the invention of the present application is shown below.

JP2005-204621 JP2004-033199 Table 2006-112327 Japanese Patent Application 2008-292285

Song X.J. et al., (2007) Nature Genet. 39, 623-630 Fan C. et al., (2006) Theor. Appl. Genet. 112, 1164-1171 Shomura A et al. (2008) Nat. Genet. 40, 1023-1028.

  The present invention has been made in view of such circumstances, and an object of the present invention is to isolate a novel gene related to the grain shape of a plant (including spikelets, fruits and seeds), and thus the elongation of the grain length. The object of the present invention is to provide a method for identification and breeding that increases the grain size of plant grains (including spikelets, fruits and seeds) using the gene.

  The inventors have so far attempted to search for genes responsible for yield increase directly, that is, genes related to elongation of grain length, using rice, which is a model of monocotyledonous plants, in order to increase crop yield of plants. The grain length is governed as a quantitative trait by the interaction of multiple genes.

  The inventors first performed linkage analysis using backcross progenies of Koshihikari and a near-isogenic line NIL (GL5) in which Koshihikari was used as a genetic background and the GL5 region was replaced with a Kasaras chromosome. As a result of linkage analysis using the SNP marker near the GL5 locus, the GL5 locus was identified between the SNP marker G1422 and the SNP marker G1491 in the range of about 7 kbp (FIG. 1). Next, the gene prediction database published by the Rice Annotation Project narrows down the GL5 gene to one gene (Os05g0187000) from the gene expression level and expression site, and this gene encodes the β2 subunit of the 20S proteasome Became clear (Figure 2).

  As a result of cDNA sequence analysis of Koshihikari and Kasalath, there were two SNPs in the coding region of the GL5 candidate gene (OsPBB1), but these did not cause any change in the amino acid sequence. The amino acid sequences of the candidate genes were found to be identical (Figure 3). It was also revealed that several mutations exist in the putative promoter region of OsPBB1 (Fig. 4).

  Next, when the expression level of the OsPBB1 gene was compared, it was revealed that the expression level of NIL (GL5) was about 2.8 times higher than that of Koshihikari (FIG. 5).

  Next, when the function of OsPBB1 was demonstrated by a transformant that overexpresses OsPBB1 in Nipponbare, which has an OsPBB1 expression level equivalent to that of Koshihikari, it was found that overexpression of OsPBB1 significantly increased the length of brown rice. (Fig. 6).

  Since high expression of GL5 gene (OsPBB1) was found to increase the grain length of rice, the effect of high expression of this gene in other plant species was further investigated. As a result, it was revealed that high expression of OsPBB1 increases seed size even in Arabidopsis thaliana (FIG. 8). This suggests that GL5 has the effect of increasing seed size not only in rice, which is a monocotyledonous plant, but also in Arabidopsis, a dicotyledonous plant, and has the same effect in a wide range of plant species.

  The GL5 gene in the present invention was identified for the first time as a gene that improves yield characteristics through grain length and grain length elongation, and can be used to increase yield and improve productivity by using the GL5 gene. That is, the present inventors succeeded in isolating a new gene involved in the increase in the grain shape of the plant body.

More specifically, the present invention provides the following [1] to [19].
[1] A plant having an increased size of seed (including seeds, spikelets or fruits) containing the DNA according to any one of (a) to (d) below.
(A) DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 6;
(B) DNA comprising a coding region of the base sequence described in SEQ ID NO: 4 or 5 and a promoter region of the base sequence described in SEQ ID NO: 1,
(C) 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: 6, or (d) SEQ ID NO: 4 Or a DNA that hybridizes under stringent conditions with the DNA comprising the nucleotide sequence according to 5
[2] The plant according to [1], wherein the plant is a monocotyledonous plant or a dicotyledonous plant.
[3] A vector introduced so that the DNA according to any one of (a) to (d) below is expressed.
(A) DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 6;
(B) DNA comprising a coding region of the base sequence described in SEQ ID NO: 4 or 5 and a promoter region of the base sequence described in SEQ ID NO: 1,
(C) 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: 6, or (d) SEQ ID NO: 4 Or a DNA that hybridizes under stringent conditions with the DNA comprising the nucleotide sequence according to 5
[4] A host cell into which the vector according to [3] has been introduced.
[5] A plant cell into which the vector according to [4] has been introduced.
[6] A transformed plant comprising the plant cell according to [5].
[7] A transformed plant that is a descendant or clone of the transformed plant according to [6].
[8] A propagation material for the transformed plant according to [6] or [7].
[9] Size of plant grains (including seeds, spikelets or fruits), comprising the step of overexpressing the DNA according to any one of (a) to (d) below in the cells of the plant body How to increase.
(A) DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 6;
(B) DNA comprising a coding region of the base sequence described in SEQ ID NO: 4 or 5 and a promoter region of the base sequence described in SEQ ID NO: 1,
(C) 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: 6, or (d) SEQ ID NO: 4 Or a DNA that hybridizes under stringent conditions with the DNA comprising the nucleotide sequence according to 5
[10] The method according to [9], wherein the plant body is a monocotyledonous plant or a dicotyledonous plant.
[11] A child of a test plant characterized by detecting a DNA marker containing a mutation present in a site corresponding to the DNA region described in SEQ ID NO: 1 or a peripheral sequence of the test plant A method for detecting the yield of fruits (including seeds, spikelets or fruits).
[12] The method according to [11], wherein the mutation is a single nucleotide polymorphism.
[13] The method according to [11], comprising the following steps (a) and (b).
(A) The base type of the polymorphic site present in the site corresponding to the coding region of the base sequence described in SEQ ID NO: 1 and the promoter region of the base sequence described in SEQ ID NO: 1 or the surrounding sequence in the test plant And (b) in the base type of the polymorphic site determined in (a), when an allele different from SEQ ID NO: 1 or its surrounding sequence is detected, the test plant is a seed (seed (14), the polymorphic site is located at positions 32, 129, 161, 557, 839 in the nucleotide sequence set forth in SEQ ID NO: 1. [13] The method according to [13], which is a site corresponding to at least one polymorphic site selected from positions 843, 976, or 1699.
[15] The mutation of the base type at the polymorphic site is a mutation from the G-to-T base position at position 32 in the base sequence described in SEQ ID NO: 1, a mutation from the A-to-G base position at position 129, 161 Mutation of the base species at position T to G, mutation of the base species at position 557 from T to C, deletion of T at the position of 839 base, deletion of G at the position of base 843, base type at position 976 In the case of at least one mutation selected from a C to A mutation or a C to T mutation of the base species at position 1699, the grain (including seeds, spikelets or fruits) is increased. The method according to [14], wherein the determination is performed.
[16] Grains of seeds (seed, spikelets or fruits) that specifically hybridize with the DNA of SEQ ID NO: 1 under stringent conditions and contain an oligonucleotide having a chain length of at least 15 nucleotides For detecting the yield of
[17] The primer according to [16] for amplifying a region containing the polymorphic site according to [14] or [15].
[18] Grains of seeds (seed, spikelets or Probe for detecting the yield of fruits (including fruit).
[19] The probe according to [18], which specifically hybridizes to a region containing the polymorphic site according to [14] or [15].

  Through linkage analysis, we succeeded in isolating and identifying genes related to the growth of plant grains (including seeds, spikelets or fruits) and thus the yield. Moreover, the breeding method which increases the length of the seed grain (a seed, a bud, or a fruit) using this gene was also discovered. The present invention is useful in fields such as plant breed improvement.

It is a figure which shows the gene specific result of QTL which concerns on grain length. It is a figure which shows the phylogenetic tree of the gene similar to GL5. It is a figure which shows the amino acid sequence comparison of GL5 gene of Koshihikari and Kasalath. It is a figure which shows the promoter sequence comparison (first half part) of GL5 gene of Koshihikari and Kasalath. It is a figure which shows the promoter sequence comparison (latter part) of GL5 gene of Koshihikari and Kasalath. It is a graph which shows the comparison of the expression level of GL5 gene in Koshihikari and NIL (GL5). It is a figure which shows the brown rice grain size of the transformant rice which overexpressed GL5 gene in Nipponbare. It is a figure which shows the amino acid sequence comparison of the gene similar to GL5 of a rice and Arabidopsis thaliana. It is a figure which shows the seed grain size of the transformed Arabidopsis thaliana which overexpressed GL5 gene in Arabidopsis thaliana.

  The present invention relates to a plant containing the gene GL5 relating to the yield of seeds (including seeds, spikelets or fruits) of the plant, and grain yield (seed, spikelets or fruits) using the gene. A method for increasing the seed yield, a method for detecting the grain yield, and a method for breeding the plant body in which the grain yield increases.

  In the present invention, the increase in grain yield is specifically brought about by increasing seeds, spikelets or fruits. In the present invention, the increase in grain means that the size of the grain increases, compared to the grain of the plant body in which the expression level of the gene is low, each 30% or more, preferably 20% or more, More preferably, it means an increase in size of 15%, 10%, 5% or more.

  The base sequence of the genomic DNA of the GL5 gene of rice casalas, the base sequence of the cDNA of which is clarified by the present inventors in relation to the grain yield of the crop, is shown in SEQ ID NO: 5, and these genes are The amino acid sequence of the encoded protein is shown in SEQ ID NO: 6.

  The nucleotide sequence of the genomic DNA of the GL5 gene of Yokoshihikari is shown in SEQ ID NO: 1, the nucleotide sequence of cDNA is shown in SEQ ID NO: 2, and the amino acid sequence of the protein encoded by these genes is shown in SEQ ID NO: 3.

  The present invention provides a vector containing a GL5 gene derived from rice having a function of increasing the grain of a plant body, and a transformed plant cell.

Specifically, the GL5 gene of the present invention includes the DNA described in any of (a) to (d) below, which encodes a rice-derived protein having a function of increasing the grain of a plant body. .
(A) DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 6;
(B) DNA comprising a coding region of the base sequence described in SEQ ID NO: 4 or 5 and a promoter region of the base sequence described in SEQ ID NO: 1,
(C) 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: 6, or (d) SEQ ID NO: 4 Or a DNA that hybridizes under stringent conditions with the DNA comprising the nucleotide sequence according to 5

  In the present invention, the plant from which the gene of the present invention is derived is not particularly limited. For example, rice, wheat, barley, oats, corn, pearl barley, Italian ryegrass, perennial ryegrass, timothy, meadow fescue, millet, millet, sugar cane Cereals such as soybeans, peas and peanuts, so-called fruit vegetables such as pumpkins and tomatoes, oil crops such as sesame, rapeseed and sunflower, Arabidopsis and the like. More preferably, rice can be mentioned as a plant from which the gene of the present invention is derived.

  The “GL5 gene” of the present invention is not particularly limited as long as it can encode a “GL5 protein”. The “GL5 gene” includes genomic DNA, chemically synthesized DNA, etc. in addition to cDNA. It is. In addition, as long as the GL5 gene encodes the GL5 protein, DNA having an arbitrary base sequence based on the degeneracy of the genetic code is included.

  Examples of the coding region of the gene of the present invention include a region at positions 217 to 1035 in the base sequence described in SEQ ID NO: 5.

  Preparation of genomic DNA and cDNA can be performed by those skilled in the art using conventional means. For genomic DNA, for example, genomic DNA is extracted from a plant, a genomic library (a vector such as a plasmid, phage, cosmid, BAC, PAC, etc. can be used) is developed, and the GL5 gene (for example, The clones can be obtained and prepared by colony hybridization or plaque hybridization using a probe prepared based on the DNA described in SEQ ID NO: 4 or 5. It is also possible to prepare a primer specific for the GL5 gene and perform PCR using this primer. In addition, for example, cDNA is synthesized based on mRNA extracted from a plant, inserted into a vector such as λZAP to create a cDNA library, developed, and colony hybridization in the same manner as described above. Alternatively, it can be prepared by performing plaque hybridization or by performing PCR.

  Furthermore, since the GL5 gene is considered to be widely present in cereal plants, the GL5 gene includes homologous genes present in various plants. Here, the “homologous gene” refers to a gene encoding a protein functionally equivalent to the active GL5 gene product in rice in various plants. Examples of such proteins include, but are not limited to, active GL5 protein mutants, alleles, variants, homologs, partial peptides of active GL5 protein, or fusion proteins with other proteins. .

  The mutant of the active GL5 protein in the present invention is a naturally occurring protein consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 6. A protein functionally equivalent to the N-terminal active fragment of the protein consisting of the amino acid sequence set forth in SEQ ID NO: 6 can be mentioned. A protein encoded by naturally-occurring DNA that hybridizes under stringent conditions with a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 4 or 5, and comprising the amino acid sequence set forth in SEQ ID NO: 6. Proteins functionally equivalent to the N-terminal active fragment of the protein can also be mentioned as mutants of the active GL5 protein.

  In addition, as a mutant of the active GL5 protein in the present invention, a naturally derived amino acid sequence consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence set forth in SEQ ID NO: 6. A protein that is functionally equivalent to the protein having the amino acid sequence set forth in SEQ ID NO: 6 can be exemplified. A protein encoded by naturally-occurring DNA that hybridizes under stringent conditions with a DNA comprising the nucleotide sequence set forth in SEQ ID NO: 4 or 5, and comprising the amino acid sequence set forth in SEQ ID NO: 6. Proteins functionally equivalent to the protein can also be mentioned as mutants of the active GL5 protein.

  In the present invention, the number of amino acids to be mutated is not particularly limited, but is usually within 30 amino acids, preferably within 15 amino acids, and more preferably within 5 amino acids (for example, within 3 amino acids). The amino acid residue to be mutated is preferably mutated to another amino acid whose amino acid side chain properties are conserved (this is known as a conservative amino acid substitution). For example, amino acid side chain properties include hydrophobic amino acids (A, I, L, M, F, P, W, Y, V) and hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T) can be broadly classified. In addition, based on the structure of the side chain, an amino acid having an aliphatic side chain (G, A, V, L, I, P), an amino acid having a hydroxyl group-containing side chain (S, T, Y), containing a sulfur atom Amino acids with side chains (C, M), amino acids with carboxylic acid and amide-containing side chains (D, N, E, Q), amino acids with base-containing side chains (R, K, H), aromatic-containing side Amino acids can also be classified such as amino acids having chains (H, F, Y, W). Furthermore, it is also well known to classify amino acids, for example, by a mutational matrix (Taylor 1986, J, Theor. Biol. 119, 205-218; Sambrook, J. et al., Molecular Cloning 3rd ed. A7 .6-A7.9, Cold Spring Harbor Lab. Press, 2001). This classification is summarized below: aliphatic amino acids (L, I, V), aromatic amino acids (H, W, Y, F), charged amino acids (D, E, R, K, H), positively charged amino acids ( R, K, H), negatively charged amino acids (D, E), hydrophobic amino acids (H, W, Y, F, M, L, I, V, C, A, G, T, K), polar amino acids ( T, S, N, D, E, Q, R, K, H, W, Y), small amino acids (P, V, C, A, G, T, S, N, D), small amino acids (A, G, S) and large (non-small) amino acids (Q, E, R, K, H, W, Y, F, M, L, I) are included (the parentheses indicate the single letter of amino acids) .

  It is already known that a polypeptide having an amino acid sequence modified by deletion, addition and / or substitution by one or more amino acid residues to a certain amino acid sequence maintains its biological activity. Yes. Furthermore, the target amino acid residue is more preferably mutated to an amino acid residue having as many common properties as possible.

  In the present invention, “functionally equivalent” means that the target protein has a biological function or biochemical function equivalent to that of the active GL5 protein. In the present invention, examples of the biological function and biochemical function of the active GL5 protein include a function of increasing seeds of grains. Biological properties include the specificity of the site to be expressed and the expression level.

  Methods well known to those skilled in the art for isolating homologous genes include hybridization techniques (Southern, EM, Journal of Molecular Biology, Vol. 98, 503, 1975) and polymerase chain reaction (PCR) techniques (Saiki). , RK, et al. Science, vol. 230, 1350-1354, 1985, Saiki, RK et al. Science, vol. 239, 487-491, 1988). That is, for those skilled in the art, an oligonucleotide that specifically hybridizes to the GL5 gene using a nucleotide sequence of the GL5 gene (for example, the DNA described in SEQ ID NO: 4 or 5) or a part thereof as a probe. As a primer, it is usually possible to isolate a homologous gene of GL5 gene from various plants.

  In order to isolate DNA encoding such a homologous gene, a hybridization reaction is usually carried out under stringent conditions. Those skilled in the art can appropriately select stringent hybridization conditions. One example is pretreatment overnight at 42 ° C. in a hybridization solution containing 25% formamide, 50% formamide under more severe conditions, 4 × SSC, 50 mM Hepes pH 7.0, 10 × Denhardt's solution, 20 μg / ml denatured salmon sperm DNA. After hybridization, a labeled probe is added and hybridization is performed by incubating overnight at 42 ° C. The cleaning solution and temperature conditions for the subsequent cleaning are about 1xSSC, 0.1% SDS, 37 ° C, more severe conditions are about 0.5xSSC, 0.1% SDS, 42 ° C, and more severe conditions are about 0.2xSSC. , 0.1% SDS, about 65 ° C. ”. Thus, isolation of DNA having high homology with the probe sequence can be expected as the conditions for washing hybridization are more severe. However, combinations of the above SSC, SDS, and temperature conditions are exemplary, and those skilled in the art will understand the above or other factors that determine the stringency of hybridization (eg, probe concentration, probe length, hybridization reaction). It is possible to achieve the same stringency as above by appropriately combining the time and the like.

  The homology of the isolated DNA is at least 50% or more, more preferably 70% or more, more preferably 90% or more (for example, 95%, 96%, 97%, 98%, 99%) in the entire amino acid sequence. And the like). Sequence homology can be determined using BLASTN (nucleic acid level) and BLASTX (amino acid level) programs (Altschul et al. J. Mol. Biol., 215: 403-410, 1990). The program is based on 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). Yes. When analyzing a base sequence by BLASTN, parameters are set to score = 100 and wordlength = 12, for example. Further, when the amino acid sequence is analyzed by BLASTX, the parameters are, for example, score = 50 and wordlength = 3. When the amino acid sequence is analyzed using the Gapped BLAST program, it can be performed as described in Altschul et al. (Nucleic Acids Res. 25: 3389-3402, 1997). When using BLAST and Gapped BLAST programs, the default parameters of each program are used. Specific methods of these analysis methods are known.

  Examples of the vector of the present invention include a vector for expressing the DNA of the present invention in a cell for producing a transformant, for example, a vector for expressing the DNA of the present invention in a plant cell for preparing a transformed plant. included. 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, a plasmid, a phage, or a cosmid can be exemplified.

  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 DNA of the present invention.

  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 or expressing the DNA of the present invention in a host cell.

  The DNA in the present invention is usually carried (inserted) into an appropriate vector and introduced into a host cell. That is, the present invention provides a host cell carrying the DNA or vector of the present invention. 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 introducing and expressing the DNA of the present invention into a cell having an endogenous gene. The expression vector is not particularly limited as long as it is a vector that expresses DNA in vitro, in E. coli, in cultured cells, or in an individual organism. For example, in the case of in vitro expression, pBEST vector (manufactured by Promega), E. coli PET vector (manufactured by Invitrogen) if present, pME18S-FL3 vector (GenBank Accession No. AB009864) for cultured cells, pME18S vector (Mol Cell Biol. 8: 466-472 (1988)) for individual organisms, plants If it is an individual, a pBINPLUS vector (van Engelen, FA et al., (1995). PBINPLUS: an improved plant transformation vector based on pBIN19. Transgenic Res. 4, 288-290.) May be exemplified. The DNA of the present invention can be inserted into a vector by a conventional method (Molecular Cloning, 5.61-5.63), 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 the cells for expressing the DNA 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.

  In addition, as a method for expressing the DNA of the present invention in vivo, the DNA of the present invention is incorporated into an appropriate vector, for example, polyethylene glycol method, electroporation method, Agrobacterium method, liposome method, cationic liposome method. , Calcium phosphate precipitation method, electric pulse perforation method (electroporation) (Current protocols in Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley & Sons. Section 9.1-9.9), lipofection method (GIBCO-BRL) ), A method of introducing into a living body by a method known to those skilled in the art, such as a microinjection method and a particle gun method.

  Administration into a plant may be an ex vivo method or an in vivo method.

  In addition, when the DNA of the present invention is introduced into a plant body, the DNA can be directly introduced into a plant cell using a microinjection method, an electroporation method, a polyethylene glycol method, etc. It can also be introduced into a plant cell indirectly via a virus or bacterium capable of infecting a plant as a vector. Examples of such viruses include cauliflower mosaic virus, tobacco mosaic virus, gemini virus and the like as typical viruses, and Agrobacterium and the like as bacteria. When introducing a gene into a plant by the Agrobacterium method, a commercially available plasmid can be used. As a method for introducing the DNA of the present invention into a plant body using such a vector, the leaf disk method (Jorgensen, RA et al., (1996), preferably introducing a gene via Agrobacterium. ). Chalcone synthase cosuppression phenotypes in petunia flowers: comparison of sense vs. antisense constructs and single-copy vs. complex T-DNA sequences. Plant Mol. Biol. 31, 957-973.).

  These transformation methods described above are preferably selected as appropriate according to the type of plant or the like that serves as the host (for example, a monocotyledonous plant or a dicotyledonous plant).

  In the present invention, `` plant '' is not particularly limited, for example, grains such as rice, wheat, barley, oat, corn, pearl barley, Italian ryegrass, perennial ryegrass, timothy, meadow fescue, millet, millet, sugar cane, soybeans, peas, Examples include beans such as peanuts, so-called fruit vegetables such as pumpkins and tomatoes, oil crops such as sesame, rapeseed and sunflower, and Arabidopsis.

  The present invention also provides a plant cell carrying the DNA of the present invention or the vector of the present invention. Furthermore, this invention provides the transformed plant body containing the plant cell of this invention. The cells into which the DNA of the present invention or the vector of the present invention is introduced include plant cells for producing transformed plants. There is no restriction | limiting in particular as a plant cell.

  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.

  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, with regard to the technique for producing a transformed plant body, a method for regenerating a plant body by introducing a gene into protoplasts using polyethylene glycol, a method for regenerating a plant body by introducing a gene into protoplasts using an electric pulse, or a gene for cells by a particle gun method. Can be mentioned, and a method for regenerating a plant body and a method for regenerating a plant body by introducing a gene via Agrobacterium are not particularly limited. Some techniques have already been established and are widely used in the technical field of the present invention. 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 neomycin phosphotransferase gene that is resistant to the antibiotics kanamycin or gentamicin, the hygromycin phosphotransferase gene that is resistant to hygromycin, and the acetyl that is 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, Fujimura et al. (Plant Tissue Culture Lett. 2:74 (1995)) can be used for rice, and Shillito et al. (Bio / Technology 7) for maize. : 581 (1989)) and Gorden-Kamm et al. (Plant Cell 2: 603 (1990)).

  In addition, the presence of introduced foreign DNA in a transformed plant that has been regenerated and cultivated in this way can be analyzed by a known PCR method or Southern hybridization method, or by analyzing the base sequence of the DNA in the plant body. Can be confirmed.

  In this case, extraction of DNA from the transformed plant body can be performed according to a known method of J. Sambrook et al. (Molecular Cloning, 2nd edition, Cold Spring Harbor Laboratory Press, 1989).

  When the foreign gene comprising the DNA of the present invention present in the regenerated plant body is analyzed using the PCR method, the amplification reaction is performed using the DNA extracted from the regenerated plant body as a template as described above. In addition, an amplification reaction is carried out in a reaction mixture in which the DNA of the present invention or a synthesized oligonucleotide having a base sequence appropriately selected according to the base sequence of the DNA modified according to the present invention is used as a primer. You can also In the amplification reaction, DNA denaturation, annealing, and extension reactions are repeated several tens of times to obtain an amplification product of a DNA fragment containing the DNA sequence of the present invention. When the reaction solution containing the amplification product is subjected to, for example, agarose electrophoresis, it is possible to confirm that the amplified DNA fragments are fractionated and the DNA fragments correspond to the DNA of the present invention.

  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 plant cells into which the DNA or vector of the present invention has been introduced, plant bodies containing the cells, progeny and clones of the plant bodies, and propagation materials for the plant bodies, their progeny, and clones.

  Plants produced in this way are expected to increase grain yield compared to normal grains.

  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 plant body or its seeds with increased grain yield of the present invention 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, it is possible to produce a plant body with increased grain or its seed.

  When the plant body or seed of the present invention is produced by the breeding method, it can be appropriately carried out with reference to various known literatures (cell engineering separate volume, plant cell engineering series 15 “Experimental protocol for model plants” Junsha, 2001, Kato Koji, “2.1 Mating of Rice and Wheat”, p.6-9; Takahiro Retsuro, Sano, Yoshio “2. Mating Method”, p.46-48).

A preferred embodiment of the breeding method of the present invention is a method including the steps described in the following (a) and (b).
(A) producing a variety in which a plant having increased seeds and a plant having an arbitrary function are crossed,
(B) Detecting the yield of the plant seed produced in step (a)

As a more specific example of the method for breeding the plant body of the present invention, a method including the steps described in the following (a) and (b) can be mentioned.
(A) a 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 for producing a plant body of the present invention is carried out by a breeding method, a method including the following steps can be mentioned.
(A) crossing plant A with other plant B having the DNA of the present invention to produce F1;
(B) crossing the F1 and the plant A,
(C) selecting a plant having the DNA,
(D) Crossing the plant selected in step (c) with the plant A

  In the above method, a plant B having the DNA of the present invention is crossed with a plant for which grain yield is to be increased or a plant for which grain is to be increased (these plants are referred to as “plant A”). The DNA of the present invention possessed by B is intentionally selected by selecting individuals that are inherited by the DNA of the present invention and having close proximity to the plant A, and then performing a “backcross” in which the crossing by the plant A is repeated. Introduce. 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”.

  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.

  The present inventors have found that rice having a GL5 gene derived from Kasalath has a function of increasing seeds. Therefore, the seed yield of the test plant can be detected by discriminating between the Koshihikari type and the Kasalath type GL5 gene. In other words, it can be used in the above breeding method by using a Kasalath type mutation site for Koshihikari type as a DNA marker.

  Based on the above findings, the present invention relates to the DNA region described in SEQ ID NO: 1 (for example, the coding region of the base sequence described in SEQ ID NO: 1 and the promoter of the base sequence described in SEQ ID NO: 1) for the test plant. The present invention provides a method for detecting seed yield of a test plant, which comprises detecting a DNA marker comprising a mutation corresponding to a site corresponding to a region) or a peripheral sequence thereof.

  In the present invention, the “corresponding site” in the DNA region described in SEQ ID NO: 1 refers to a corresponding site in a homologous gene of the above gene present in the plant in various plants.

  In the present invention, “detecting grain yield” includes a test for determining whether or not the grain size increases. In the method of the present invention, when a mutation of the same type as SEQ ID NO: 4 is detected at the site corresponding to the DNA region described in SEQ ID NO: 1 or its peripheral sequence, the size of the seed increases. Then, it is determined.

  By detecting the mutation in the test plant by the method of the present invention, it is possible to determine whether or not the grain size of the test plant increases without actually observing the grain.

  The “peripheral sequence” in the present invention usually refers to a region on the chromosome in the vicinity of the gene. The vicinity is not particularly limited, but is usually a DNA region containing the polymorphic site of the present invention.

  The position of the “mutation” in the detection method of the present invention is difficult to pre-define, but is usually in the ORF of the gene or a region that controls the expression of the gene (eg, promoter region, enhancer region, etc.) ), But is not limited thereto. In addition, the “mutation” is a mutation that changes the expression level of the gene, changes properties such as mRNA stability, or changes the activity of the protein encoded by the gene. Many, but not particularly limited. Examples of the mutation of the present invention include base addition, deletion, substitution, insertion mutation and the like.

  In the DNA region of SEQ ID NO: 1, which encodes a plant-derived protein having a function of increasing plant grain yield in the test plant, the present inventors are significantly related to the plant grain yield. We succeeded in finding polymorphic mutations.

  The “polymorphic site” in the method for detecting grain yield of the present invention is not particularly limited as long as it is a polymorphism present in the base sequence described in any of the above DNAs or a DNA region in the vicinity of the base sequence. Specifically, polymorphic sites that can be used in the method for detecting grain yield of the present invention include positions 32, 129, 161, 557, 839, 843 in the base set forth in SEQ ID NO: 1. , Position corresponding to at least one polymorphic site selected from position 976 or 1699 (in the present specification, these polymorphic sites are simply referred to as “polymorphic sites of the present invention”). May be listed).

  The base type of the polymorphic site shown in the above table may indicate a base type on the complementary strand side with respect to the sequence shown in the sequence table, but if the sequence before and after described in this specification is used, It is easy for those skilled in the art to confirm the difference, and when performing detection, the other result can be determined inevitably by examining either the plus strand or the minus strand.

  In a preferred embodiment of the present invention, the mutation of the base species at the polymorphic site described above is a mutation from G to T of the base species at position 32 in the base sequence set forth in SEQ ID NO: SEQ ID NO: 1, base at position 129 Species A to G mutation, 161th base type T to G mutation, 557th base type T to C mutation, 839th base type T deletion, 843rd base type The seed is determined to increase when the G is deficient, at least one mutation selected from the C-to-A mutation of the base species at position 976, or the C-to-T mutation at position 1699 Is done.

  As described above, according to the present invention, a region on a gene related to grain yield has been clarified, so that the grain yield can be detected without imposing an excessive burden on those skilled in the art.

  A person skilled in the art can determine the base species in the polymorphic site of the present invention by various methods. For example, it can be performed by directly determining the base sequence of DNA containing the polymorphic site of the present invention.

  The “test plant” to be subjected to the detection method of the present invention is not particularly limited, but preferably includes rice, more preferably rice Koshihikari varieties and Kasalath varieties.

  In general, the test sample used in the detection method of the present invention is preferably a biological sample obtained in advance from a test plant. Examples of the biological sample include a DNA sample. The DNA sample in the present invention can be prepared based on, for example, chromosomal DNA extracted from tissues or cells of a test plant, or RNA.

  That is, the present invention is a method in which a biological sample derived from a subject (a biological sample obtained in advance from a test plant) is used as a test sample.

  A person skilled in the art can appropriately prepare a biological sample using a known technique. For example, a DNA sample can be prepared by PCR using a primer that hybridizes to DNA containing the polymorphic site of the present invention and chromosomal DNA or RNA as a template.

  In this method, the base sequence of the isolated DNA is then determined. Those skilled in the art can easily determine the base sequence of the isolated DNA using a DNA sequencer or the like.

  Various methods for determining the base type of a polymorphic site whose base variation has been clarified in advance are known. The method for determining the base species of the present invention is not particularly limited. For example, TaqMan PCR method, AcycloPrime method, MALDI-TOF / MS method and the like have been put to practical use as analysis methods applying the PCR method. Invader and RCA methods are known as methods for determining base types that do not depend on PCR. Furthermore, the base species can be determined using a DNA array. Any of the methods described herein can be applied to the determination of the base type of the polymorphic site in the present invention.

  All of these methods have been developed for genotyping a large amount of samples at high speed. With the exception of MALDI-TOF / MS, it is usually necessary to prepare a labeled probe or the like in any way for either method. On the other hand, a method for determining a base type that does not rely on a labeled probe has been performed for a long time. As one of such methods, for example, a method using restriction fragment length polymorphism (RFLP), a PCR-RFLP method and the like can be mentioned.

  RFLP utilizes the fact that a restriction enzyme recognition site mutation or a base insertion or deletion in a DNA fragment caused by restriction enzyme treatment can be detected as a change in the size of the fragment produced after the restriction enzyme treatment. If there is a restriction enzyme that recognizes the base sequence containing the polymorphism to be detected, the base of the polymorphic site can be known by the principle of RFLP.

  Alternatively, a CAPS (Cleaved Amplifeid Polymorphic Sequence) marker or a primer can be used to introduce a mutation and a dCAPS (derived CAPS) marker that creates a restriction enzyme site. The dCAPS marker is a technique of creating a restriction enzyme site on a PCR product by causing a mismatch between a PCR primer and DNA of a template (Japanese Patent Laid-Open No. 2003-259898).

  As a method that does not require a labeled probe, a method for detecting a difference in base using a change in the secondary structure of DNA as an index is also known. PCR-SSCP utilizes the fact that the secondary structure of single-stranded DNA reflects the difference in its nucleotide sequence (Cloning and polymerase chain reaction-single-strand conformation polymorphism analysis of anonymous Alu repeats on chromosome 11. Genomics 1992 Jan 1; 12 (1): 139-146., Detection of p53 gene mutations in human brain tumors by single-strand conformation polymorphism analysis of polymerase chain reaction products. Oncogene. 1991 Aug 1; 6 (8): 1313- 1318., Multiple fluorescence-based PCR-SSCP analysis with postlabeling., PCR Methods Appl. 1995 Apr 1; 4 (5): 275-282.). The PCR-SSCP method is performed by dissociating the PCR product into single-stranded DNA and separating it on a non-denaturing gel. Since the mobility on the gel varies depending on the secondary structure of the single-stranded DNA, if there is a difference in base at the polymorphic site, it can be detected as a difference in mobility.

  Other examples of methods that do not require a labeled probe include denaturant gradient gel electrophoresis (DGGE method). The DGGE method is a method in which a mixture of DNA fragments is run in a polyacrylamide gel having a concentration gradient of a denaturing agent, and the DNA fragments are separated depending on the instability of each. When a mismatched unstable DNA fragment migrates to a certain denaturant concentration in the gel, the DNA sequence around the mismatch is partially dissociated into single strands due to its instability. The mobility of a partially dissociated DNA fragment becomes very slow and can be separated from the mobility of a complete double-stranded DNA without a dissociated portion.

  In addition, the DNA species can be used to determine the base species (Cell engineering separate volume “DNA microarray and the latest PCR method”, Shujunsha, published 2000.4 / 20, pp97-103 “SNP analysis using oligo DNA chip”, Sabae. Shinichi). In the DNA array, sample DNA (or RNA) is hybridized to a large number of probes arranged on the same plane, and the hybridization to each probe is detected by scanning the plane. Since responses to many probes can be observed simultaneously, for example, a DNA array is useful for analyzing a large number of polymorphic sites simultaneously.

  In addition to the above method, an allele specific oligonucleotide (ASO) hybridization method can be used to detect a base at a specific site. An allyl-specific oligonucleotide (ASO) is composed of a base sequence that hybridizes to a region where a polymorphic site to be detected is present. When ASO is hybridized to sample DNA, the hybridization efficiency decreases if a mismatch occurs at the polymorphic site due to the polymorphism. Mismatches can be detected by Southern blotting or a method that uses the property of quenching by intercalating a special fluorescent reagent into the hybrid gap. Mismatches can also be detected by the ribonuclease A mismatch cleavage method.

  Among the oligonucleotides, at least one polymorphic site selected from the 32, 129, 161, 557, 839, 843, 976, or 1699 position in the nucleotide sequence set forth in SEQ ID NO: 1. An oligonucleotide having a chain length of at least 15 nucleotides that hybridizes to DNA containing any one of the polymorphic sites among the sites corresponding to can be used as a reagent (test agent) for detecting the fruit yield. This is used for a test using gene expression as an index or a test using gene polymorphism as an index.

  The oligonucleotide specifically hybridizes to DNA containing any one of the polymorphic sites of the present invention. Here, “specifically hybridize” means normal hybridization conditions, preferably stringent hybridization conditions (for example, Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, In the condition described in the second edition 1989), it means that cross-hybridization does not occur significantly with DNA encoding other proteins. If the specific hybridization is possible, the oligonucleotide becomes a DNA base sequence encoding a plant-derived protein having a function of increasing the fruit of the plant in the gene to be detected or a DNA region in the vicinity of the gene. On the other hand, it need not be completely complementary.

  The oligonucleotide can be used as a probe or primer in the test method of the present invention. When the oligonucleotide is used as a primer, the length is usually 15 bp to 100 bp, preferably 17 bp to 30 bp. The primer is at least one polymorphism selected from position 32, position 129, position 161, position 557, position 839, position 843, position 976 or position 1699 in the nucleotide sequence set forth in SEQ ID NO: 1 of the present invention. Of the sites corresponding to the sites, any DNA can be used as long as it can amplify at least a part of the DNA containing any polymorphic site.

  The present invention provides a primer for amplifying a region containing the polymorphic site of the present invention, and a probe that hybridizes to a DNA region containing the polymorphic site.

  In the present invention, the primer for amplifying a region containing a polymorphic site also includes a primer that can initiate complementary strand synthesis toward the polymorphic site using DNA containing the polymorphic site as a template. The primer can also be expressed as a primer for providing a replication origin on the 3 ′ side of the polymorphic site in DNA containing the polymorphic site. The interval between the region where the primer hybridizes and the polymorphic site is arbitrary. For the interval between the two, a suitable number of bases can be selected according to the analysis method of the base at the polymorphic site. For example, in the case of a primer for analysis using a DNA chip, the primer can be designed so as to obtain an amplification product having a length of 20 to 500, usually 50 to 200 bases, as a region containing a polymorphic site. A person skilled in the art can design a primer according to the analysis method based on the base sequence information about the surrounding DNA region including the polymorphic site. The base sequence constituting the primer of the present invention can be appropriately modified as well as a base sequence completely complementary to the genomic base sequence.

  In addition to the base sequence complementary to the genomic base sequence, an arbitrary base sequence can be added to the primer of the present invention. For example, in a primer for a polymorphism analysis method using a type IIs restriction enzyme, a primer to which a recognition sequence for a type IIs restriction enzyme is added is used. Such a primer with a modified base sequence is included in the primer of the present invention. Furthermore, the primer of the present invention can be modified. For example, a fluorescent substance or a primer labeled with a binding affinity substance such as biotin or digoxin is used in various genotyping methods. Primers having these modifications are also included in the present invention.

  The present invention also provides a test agent or kit for the polymorphic site of the present invention, which contains the above primer as an active ingredient.

  On the other hand, in the present invention, a probe that hybridizes to a region containing a polymorphic site refers to a probe that can hybridize to a polynucleotide having a base sequence of a region containing a polymorphic site. More specifically, a probe containing a polymorphic site in the base sequence of the probe is preferable as the probe of the present invention. Alternatively, depending on the base analysis method at the polymorphic site, the probe may be designed so that the end of the probe corresponds to a base adjacent to the polymorphic site. Therefore, although the polymorphic site is not included in the base sequence of the probe itself, a probe including a base sequence complementary to the region adjacent to the polymorphic site can also be shown as a desirable probe in the present invention.

  In other words, a probe capable of hybridizing to the polymorphic site of the present invention on genomic DNA or a site adjacent to the polymorphic site is preferred as the probe of the present invention. In the probe of the present invention, modification of the base sequence, addition of the base sequence, or modification is allowed in the same manner as the primer. For example, a probe used for the Invader method is added with a base sequence unrelated to the genome constituting the flap. Such a probe is also included in the probe of the present invention as long as it hybridizes to a region containing a polymorphic site. The base sequence constituting the probe of the present invention can be designed according to the analysis method based on the base sequence of the DNA region surrounding the polymorphic site of the present invention in the genome.

  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, usually 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.

  Specific examples of the probe of the present invention include a G-to-T mutation at position 32 in the base sequence set forth in SEQ ID NO: 1, a A-to-G mutation at position 129, 161 Mutation of the base species at position T to G, mutation of the base species at position 557 from T to C, deletion of T at the position of 839 base, deletion of G at the position of base 843, base type at position 976 A probe that hybridizes to a region containing a polymorphic site corresponding to the polymorphic site of at least one mutation selected from a C to A mutation of C1 or a mutation of C1 to T of a base species at position 1699 And a probe having a chain length of at least 15 nucleotides.

  The present invention also provides a reagent (test agent) for use in the method for detecting grain yield of the present invention. The reagent of the present invention includes the primer and / or probe of the present invention. In the detection of grain yield, a primer and / or probe for amplifying DNA containing the polymorphic site described in any of the polymorphic sites of the present invention is used.

  Various reagents, enzyme substrates, buffers and the like can be combined with the reagent of the present invention depending on the method for determining the base species. As the enzyme, enzymes necessary for various analysis methods exemplified as the above-mentioned base species determination method such as DNA polymerase, DNA ligase, or IIs restriction enzyme can be shown. As the buffer solution, a buffer solution suitable for maintaining the activity of the enzyme used for these analyzes is appropriately selected. Furthermore, as the enzyme substrate, for example, a substrate for complementary strand synthesis is used.

  Furthermore, another embodiment of the reagent in the present invention is a reagent for detecting grain yield, which comprises a solid phase on which a nucleotide probe that hybridizes with the DNA containing the polymorphic site of the present invention is immobilized.

  These are used for examinations using the polymorphic site of the present invention as an index. These preparation methods can be carried out by methods known to those skilled in the art.

  In addition, all prior art documents cited in the present specification are incorporated herein by reference.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

[Example 1] Positional cloning of GL5 gene The GL5 gene was identified by linkage analysis. The population used for the analysis used Koshihikari and Koshihikari as a genetic background and backcross progeny with a near-isogenic line NIL (GL5) in which the GL5 region was replaced with a Kasaras chromosome. Recombinant individuals in the vicinity of the GL5 locus using the single nucleotide polymorphism (SNP) marker G1358 and SNP marker G1569, which sandwich the GL5 region, from 2,000 individuals of the inbred progeny of individuals in which the GL5 region became heterozygous Selected. As a result of linkage analysis using the SNP marker near the GL5 locus, the GL5 locus was identified between the SNP marker G1422 and the SNP marker G1491 in the range of about 7 kbp (FIG. 1).

  The gene prediction database (http://rapdb.dna.affrc.go.jp/) published by the Rice Annotation Project predicted the presence of four genes between SNP marker G1422 and SNP marker G1491. . From these four genes, the GL5 gene was narrowed down to one gene (Os05g0187000) from the gene expression level and expression site. This gene encoded the β2 subunit of the 20S proteasome. There is only one gene (OsPBB1) in rice that encodes the β2 subunit of the 20S proteasome (Figure 2).

[Example 2] Nucleotide sequence and expression analysis of candidate gene As a result of cDNA sequence analysis of Koshihikari and Kasalath, two SNPs [position 33 (position 976 in SEQ ID NO: 1) at the coding region of GL5 candidate gene (OsPBB1) C) was A, and C at position 267 (corresponding to position 1699 of SEQ ID NO: 1) had T]. The amino acid sequences of the candidate genes were identical (Figure 3).

  In the putative promoter region (1 kbp) of OsPBB1, there are three SNPs [G at position -912 (corresponding to position 32 of SEQ ID NO: 1) T, position -815 (corresponding to position 129 of SEQ ID NO: 1) A is G, T at position −783 (corresponding to position 161 of SEQ ID NO: 1) is G], and a single base deletion insertion is missing at 2 positions [−105 (corresponding to position 839 of SEQ ID NO: 1)] , -101 position (corresponding to position 843 of SEQ ID NO: 1) was deleted] (FIGS. 4-1 and 4-2). Therefore, the expression level of the OsPBB1 gene was analyzed. Young panicles with a total length of 10 cm were collected from Koshihikari and NIL (GL5), and cDNA was obtained by the AMV reverse transcriptase reaction of WAKO using total RNA extracted by the acidic phenol method as a template. Using the obtained cDNA as a template, the expression level of the OsPBB1 gene was compared by a real-time PCR method using primers (5′-GGCAATTCGTGGTGGTATCT-3 ′, SEQ ID NO: 9 and 5′-ACCTTCGGCTTCAGTTGTGT-3 ′, SEQ ID NO: 10). . As a result, the expression level of NIL (GL5) was about 2.8 times higher than that of Koshihikari (FIG. 5).

[Example 3] Function verification of GL5 candidate gene (OsPBB1)
Since the expression level of OsPBB1 was thought to determine the grain length of Koshihikari and NIL (GL5), the function of OsPBB1 was verified by a transformant that overexpresses OsPBB1 in Nipponbare, which has the same OsPBB1 expression level as Koshihikari. .

  A transformant (BM349) in which OsPBB1 full-length cDNA (AK103126) was ligated between maize-derived ubiquitin 1 promoter and nopaline synthase terminator and overexpressed was transformed into rice FOX hunting system (Nakamura et al. (2007) Plant Mol. Biol. 65, 357-371). The solid seeds harvested from this transformant T2 generation plant were dried at 25 degrees for 2 weeks, and then the inner and outer pods were removed to obtain brown rice. The grain length of this brown rice increased significantly (P = 0.0039), and overexpression of OsPBB1 significantly increased the length of brown rice (FIG. 6). This proved that OsPBB1 is a GL5 gene.

[Example 4] Effect of GL5 on other plant species
Since the high expression of GL5 gene (OsPBB1) was found to increase the grain length of rice, the effect of high expression of this gene in other plant species was examined. Arabidopsis thaliana has two genes, AtPBB1 (SEQ ID NO: 11) and AtPBB2 (SEQ ID NO: 12), as homologous genes for OsPBB1, both of which have high homology of 82.7% with OspBB1. It has homology (Figs. 2 and 7). Therefore, we hypothesized that high expression of rice-derived OsPBB1 would increase seed size even in Arabidopsis.

  Primer (5'-CGCATCTAGACCGCGCGGGATGGCCGGAGC-3 ', SEQ ID NO: 13) ligated to the gene coding region of rice-derived OsPBB1 full-length cDNA (AK103126) and SacI site (5'-GTAAGAGCTCAAAATCACTCCTCCATTGCA-3' , SEQ ID NO: 14), and the amplified product was treated with XbaI and SacI to obtain an insert. This insert was ligated into the XbaI and SacI sites of the pSTARH301G binary vector to obtain a binary vector (pSTARH301G / GL5) in which the full-length OsPBB1 cDNA was ligated between the cauliflower mosaic virus-derived 35S promoter and the nopaline synthase terminator. . pSTARH301G / GL5 was introduced into Agrobacterium (Agrobacterium tumefaciens EHA101) by electroporation, and further Agrobacterium was infected into Arabidopsis thaliana by reduced pressure infiltration method to obtain transformant T1 seeds. This seed was subjected to transformation screening on a GM agar medium containing 50 mg / l hygromycin. T2 seeds were obtained from the obtained transformants (T1 generation), sown again in a medium containing hygromycin, a line showing a 3: 1 segregation ratio was selected, and the transgene is considered to exist at one locus. Transformants obtained were selected. When the seed size was measured, the major axis of the seed was significantly increased as compared with the control vector line not containing the cDNA of OsPBB1 (FIG. 8). High expression of OsPBB1 was found to increase seed size even in Arabidopsis. This suggests that GL5 has the effect of increasing seed size not only in rice, which is a monocotyledonous plant, but also in Arabidopsis, a dicotyledonous plant, and has the same effect in a wide range of plant species.

Claims (19)

The plant body which increased the magnitude | size of the seed of a plant body (a seed, a flower, or a fruit is included) containing the DNA in any one of (a) to (d) below.
(A) DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 6;
(B) DNA comprising a coding region of the base sequence described in SEQ ID NO: 4 or 5 and a promoter region of the base sequence described in SEQ ID NO: 1,
(C) 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: 6, or (d) SEQ ID NO: 4 Or a DNA that hybridizes under stringent conditions with the DNA comprising the nucleotide sequence according to 5   The plant body according to claim 1, wherein the plant body is a monocotyledonous plant or a dicotyledonous plant. A vector introduced so that the DNA according to any one of (a) to (d) below is expressed.
(A) DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 6;
(B) DNA comprising a coding region of the base sequence described in SEQ ID NO: 4 or 5 and a promoter region of the base sequence described in SEQ ID NO: 1,
(C) 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: 6, or (d) SEQ ID NO: 4 Or a DNA that hybridizes under stringent conditions with the DNA comprising the nucleotide sequence according to 5   A host cell into which the vector according to claim 3 has been introduced.   A plant cell into which the vector according to claim 4 is introduced.   A transformed plant comprising the plant cell according to claim 5.   A transformed plant which is a descendant or clone of the transformed plant according to claim 6.   The propagation material of the transformed plant body of Claim 6 or 7. A method for increasing the size of seeds, spikelets or fruits of a plant comprising the step of overexpressing the DNA according to any one of (a) to (d) below in the cells of the plant.
(A) DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 6;
(B) DNA comprising a coding region of the base sequence described in SEQ ID NO: 4 or 5 and a promoter region of the base sequence described in SEQ ID NO: 1,
(C) 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: 6, or (d) SEQ ID NO: 4 Or a DNA that hybridizes under stringent conditions with the DNA comprising the nucleotide sequence according to 5   The method according to claim 9, wherein the plant body is a monocotyledonous plant or a dicotyledonous plant.   About the test plant body, detecting a DNA marker containing a mutation corresponding to a site corresponding to the DNA region described in SEQ ID NO: 1 or a peripheral sequence thereof, seeds and spikelets of the test plant body Or a method for detecting the yield of fruits.   The method according to claim 11, wherein the mutation is a single nucleotide polymorphism. The method according to claim 11, comprising the following steps (a) and (b):
(A) The base type of the polymorphic site present in the site corresponding to the coding region of the base sequence described in SEQ ID NO: 1 and the promoter region of the base sequence described in SEQ ID NO: 1 or the surrounding sequence in the test plant And (b) when an allele different from SEQ ID NO: 1 or its surrounding sequence is detected in the base type of the polymorphic site determined in (a), the test plant is a seed (seed , Including a flower or a fruit)   The polymorphic site is at least one polymorphic site selected from positions 32, 129, 161, 557, 839, 843, 976, or 1699 in the nucleotide sequence set forth in SEQ ID NO: 1. The method according to claim 13, which is a corresponding site.   The mutation of the base type of the polymorphic site is a mutation from the base species G at position 32 in the base sequence described in SEQ ID NO: 1, a mutation from base A at position 129, a base at position 161 Species T to G mutation, 557 base species T to C mutation, 839 base position T defect, 843 base position G defect, 976 position base species C In the case of at least one mutation selected from the mutation to A or the mutation C to T of the base species at position 1699, it is determined that the seed (including seeds, spikelets or fruits) increases. The method according to claim 14.   Grains of test plants (including seeds, spikelets or fruits) that specifically hybridize with the DNA of SEQ ID NO: 1 under stringent conditions and contain oligonucleotides having a chain length of at least 15 nucleotides For detecting the yield of.   The primer according to claim 16 for amplifying a region containing the polymorphic site according to claim 14 or 15.   Grains (including seeds, spikelets or fruits) of the test plant, which specifically hybridizes with the DNA of SEQ ID NO: 1 under stringent conditions and contains an oligonucleotide having a chain length of at least 15 nucleotides ) Probe for detecting yield.   The probe according to claim 18, which specifically hybridizes to a region containing the polymorphic site according to claim 14 or 15.