JP5046001B2 - Genes related to the number of primary infarcts in rice and use thereof - Google Patents

Description

  The present invention relates to rice breeding, and more particularly to rice breeding in which the number of primary branch infarcts is increased.

  Rice is one of the most important foods for humanity. Rice with high yield is strongly sought around the world.

  In order to breed rice with high yield, methods such as crossing different rice lines have been taken. In recent years, molecular genetic knowledge that can be used for breeding rice with high yield has been reported.

For example, Patent Literature 1 and Non-Patent Literature 1 report genes that increase the number of rice secondary branch infarcts, respectively. As shown in FIG. 1, rice culm arrives at a primary stamen branched from the cocoon and a secondary stamen branched from the primary stamen. Accordingly, the number of rice pods into which the above gene has been introduced increases as the number of secondary branch infarcts increases.
JP 2004-89026 A (published March 15, 2004) Japanese Patent Laying-Open No. 2005-278499 (published on October 13, 2005) Cytokinin Oxidase Regulates Rice Grain Production, Motoyuki Ashikari, Hitoshi Sakakibara, Shaoyang Lin, Toshio Yamamoto, Tomonori Takashi, Asuka Nishimura, Enrique R. Angeles, Qian Qian, Hidemi Kitano, Makoto Matsuoka. (2005), SCIENCE VOL 309: 741-745 Quantitative Trait Loci for Sink Size and Ripening Traits in Rice (Oryza sativa L.), Kenji Nagata, Yoshimichi Fukuta, Hiroyuki Shimizu, Tadashi Yagi, Tomio Terao. (2002), Breeding Science VOL 52: 259-273

  However, it is generally known that cocoons that arrive at the secondary branch have poor ripening and poor quality compared to those that arrive at the primary branch. The genes described in Patent Document 1 and Non-Patent Document 1 both increase the ratio of the number of secondary branch infarcts to the number of primary branch infarcts. For this reason, in rice into which the above gene has been introduced, the proportion of culm that has reached the secondary branch in the whole harvested culm is increased, and the quality of the harvested product is degraded.

  The present invention has been made in view of the above problems, and an object thereof is to provide high-quality and high-yield rice.

  Based on a unique idea, the inventors have increased the number of primary branches without increasing the ratio of the number of secondary branches to the number of primary branches to increase the yield without reducing the quality of the harvest. Considering that the gene to be made is necessary, we are studying earnestly. So far, from the QTL analysis of the backcross line of Sasanishiki, which is a japonica-type rice cultivar, and Habataki, which is an indica-type rice cultivar, the ratio of the number of secondary branch infarcts to the number of primary branch infarcts is 1 It has been reported that a QTL that increases the number of next branch infarcts was found on the sixth chromosome (FIG. 2, Non-Patent Document 2).

It is very important to isolate the causative gene of QTL. This is because, unless the causative gene is isolated, extra traits are brought in when breeding. However, until now, it has not been possible to breed high-quality and high-yield rice using the QTL. As a result of trial and error, the present inventors have isolated the causative gene by adopting a unique technique, and have completed the present invention.
In order to solve the above problems, the polynucleotide according to the present invention is a polynucleotide comprising any one of the following base sequences (A) to (C), and the number of secondary branch infarcts relative to the number of primary branch infarcts in rice: It encodes a polypeptide that can increase the number of primary branch infarcts without increasing the ratio of: (A) the base sequence shown in SEQ ID NO: 1; (B) in the base sequence shown in SEQ ID NO: 1 A nucleotide sequence in which one or several nucleotides are substituted, added, or deleted; or (C) a nucleotide sequence capable of hybridizing under stringent conditions with a complementary sequence of the nucleotide sequence represented by SEQ ID NO: 1.

  The polynucleotide according to the present invention is a polynucleotide that encodes a polypeptide comprising any one of the following amino acid sequences (A) or (B), and has a ratio of the number of secondary branch infarcts to the number of primary branch infarcts in rice: It may encode a polypeptide that can increase the number of primary branch infarcts without increasing: (A) the amino acid sequence shown in SEQ ID NO: 3; or (B) in the amino acid sequence shown in SEQ ID NO: 3, An amino acid sequence in which one or several amino acids are substituted, added or deleted.

  The polynucleotide according to the present invention is a polynucleotide comprising any one of the following base sequences (A) to (C), and is 1 without increasing the ratio of the number of secondary branches to the number of primary branches in rice. It is preferable that it encodes a polypeptide capable of increasing the number of next branch infarction: (A) the base sequence shown in SEQ ID NO: 2; (B) one or several nucleotides in the base sequence shown in SEQ ID NO: 2 A base sequence in which is substituted, added or deleted; or (C) a base sequence capable of hybridizing with a complementary sequence of the base sequence shown in SEQ ID NO: 2 under stringent conditions.

  The polypeptide according to the present invention is characterized by being encoded by the polynucleotide according to the present invention.

  The vector according to the present invention is characterized by including the polynucleotide according to the present invention.

  The antibody according to the present invention is characterized by specifically binding to the polypeptide according to the present invention.

  The method for producing rice in which the number of primary branch infarcts has increased without increasing the ratio of the number of secondary branch infarcts to the number of primary branch infarcts according to the present invention includes the step of introducing the polynucleotide according to the present invention into rice. It is a feature.

  Rice having an increased number of primary branch infarcts without increasing the ratio of the number of secondary branch infarcts to the number of primary branch infarcts according to the present invention does not increase the ratio of the number of secondary branch infarcts to the number of primary branch infarcts according to the present invention. It is characterized by being a descendant of rice produced by the rice production method with an increased number of primary branch infarcts.

  A rice production kit in which the number of primary branch infarcts is increased without increasing the ratio of the number of secondary branch infarcts to the number of primary branch infarcts according to the present invention is characterized by comprising the polynucleotide according to the present invention.

  The rice screening method for increasing the number of primary branch infarcts without increasing the ratio of the number of secondary branch infarcts to the number of primary branch infarcts according to the present invention includes a step of incubating a rice extract with the polynucleotide according to the present invention. It is characterized by doing.

  A rice screening kit in which the number of primary branch infarcts has increased without increasing the ratio of the number of secondary branch infarcts to the number of primary branch infarcts according to the present invention is characterized by comprising the polynucleotide according to the present invention.

  The rice screening method for increasing the number of primary branch infarcts without increasing the ratio of the number of secondary branch infarcts to the number of primary branch infarcts according to the present invention includes a step of incubating a rice extract with the antibody according to the present invention. It is characterized by that.

  A rice screening kit in which the number of primary branch infarcts is increased without increasing the ratio of the number of secondary branch infarcts to the number of primary branch infarcts according to the present invention is characterized by comprising the antibody according to the present invention.

  By using the present invention, the number of primary branch infarcts can be increased without increasing the ratio of the number of secondary branch infarcts to the number of rice primary branch infarcts, so that high quality and high yield rice can be bred.

[1: Polynucleotide]
In one aspect, the present invention provides a polynucleotide encoding a polypeptide that increases the number of primary branches without increasing the ratio of the number of secondary branches to the number of primary branches in rice.

  As used herein, the term “primary branch” is intended to branch directly from rice straw. In addition, the term “secondary branch rachis” is intended to be a branch that branches off from the primary branch rachis. FIG. 1 is an explanatory diagram of rice branching.

  As used herein, the term “polypeptide” is used interchangeably with “peptide” or “protein”. In addition, “fragment” of a polypeptide is intended to be a partial fragment of the polypeptide. The polypeptides according to the invention may also be isolated from natural sources or chemically synthesized.

  The term “isolated” polypeptide or protein is intended to be a polypeptide or protein that has been removed from its natural environment. For example, recombinantly produced polypeptides and proteins expressed in a host cell can be isolated in the same manner as natural or recombinant polypeptides and proteins that have been substantially purified by any suitable technique. It is thought that there is.

  As used herein, the term “polynucleotide” is used interchangeably with “gene”, “nucleic acid” or “nucleic acid molecule” and is intended to be a polymer of nucleotides. As used herein, the term “base sequence” is used interchangeably with “nucleic acid sequence” or “nucleotide sequence” and refers to the sequence of deoxyribonucleotides (abbreviated A, G, C, and T). As shown.

  The polynucleotide according to the present invention preferably encodes a polypeptide that increases the number of primary branches without increasing the ratio of the number of secondary branches to the number of primary branches in rice. When the amino acid sequence of a specific polypeptide is obtained, the base sequence of the polynucleotide encoding the polypeptide can be easily designed.

  Further, the polynucleotide according to the present invention is more preferably a polynucleotide consisting of the base sequence shown in SEQ ID NO: 1 or 2 or a variant thereof, and a polynucleotide consisting of the base sequence shown in SEQ ID NO: 2. Is more preferable.

  As shown in the examples described later, rice having a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 1 or 2 has an increased ratio of the number of secondary branch infarcts to the number of primary branch infarcts compared to rice not having them. The number of primary branch infarcts is increasing. Also, rice having a polynucleotide consisting of the base sequence shown in SEQ ID NO: 2 is preferable because the harvest index is further improved.

  The ORF of the polypeptide according to the present invention starts from the 2296th position of the base sequence shown in SEQ ID NO: 1 and ends at the 3658th position. Therefore, the polynucleotide consisting of the base sequence represented by SEQ ID NO: 1 includes about 5 kbp upstream of the ORF region of the polypeptide of the present invention. The upstream region of the polynucleotide of the present invention is May be shorter. As shown in Examples described later, the number of primary branch infarcts also increases in rice into which a polynucleotide having a truncated upstream region has been introduced.

  The polynucleotide according to the present invention may be any polynucleotide that encodes the polypeptide according to the present invention. That is, the polynucleotide according to the present invention is a polynucleotide comprising the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 3, or the amino acid sequence shown in SEQ ID NO: 3, wherein one or several amino acids are substituted or added. Alternatively, it may be a polynucleotide consisting of a base sequence encoding a deleted amino acid sequence. As will be described later, the polypeptide according to the present invention functions to increase the number of primary branch infarcts in rice.

As used herein with respect to a polynucleotide, the term “variant” intends a polynucleotide that encodes a polypeptide that retains the same activity as that of the particular polypeptide. That is, as used herein, a variant in terms of a polynucleotide encodes a polypeptide that increases the number of primary branch infarcts without increasing the ratio of the number of secondary branch infarcts to the number of primary branch infarcts in rice. A polynucleotide comprising:
A polynucleotide comprising a base sequence in which one or several bases are substituted, deleted or added in the base sequence shown in either SEQ ID NO: 1 or 2, or any of SEQ ID NO: 1 or 2 Or a polynucleotide capable of hybridizing under stringent conditions with a complementary sequence of the base sequence shown in FIG.

  The polynucleotide according to the present invention may exist in the form of RNA (eg, mRNA) or in the form of DNA (eg, cDNA or genomic DNA). DNA can be double-stranded or single-stranded. Single-stranded DNA or RNA can be the coding strand (also known as the sense strand) or it can be the non-coding strand (also known as the antisense strand).

  As used herein, the term “oligonucleotide” is intended to be a combination of several to several tens of nucleotides, and is used interchangeably with “polynucleotide”. Oligonucleotides are called dinucleotides (dimers) and trinucleotides (trimers) as short ones, and are represented by the number of nucleotides polymerized such as 30 mers or 100 mers as long ones. Oligonucleotides can be produced as fragments of longer polynucleotides or chemically synthesized.

  The polynucleotide according to the present invention can also be fused to the polynucleotide encoding the tag tag (tag sequence or marker sequence) described above on the 5 'or 3' side.

  Hybridization is described in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2d Ed. , Cold Spring Harbor Laboratory (1989). Usually, the higher the temperature and the lower the salt concentration, the higher the stringency (harder to hybridize), and a more homologous polynucleotide can be obtained. The appropriate hybridization temperature varies depending on the base sequence and the length of the base sequence. For example, when a DNA fragment consisting of 18 bases encoding 6 amino acids is used as a probe, a temperature of 50 ° C. or lower is preferable.

  As used herein, the term “stringent hybridization conditions” refers to hybridization solution (50% formamide, 5 × SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate. (Containing pH 7.6), 5 × Denhart solution, 10% dextran sulfate, and 20 μg / ml denatured sheared salmon sperm DNA) overnight at 42 ° C., then 0.1 × at about 65 ° C. It is intended to wash the filter in SSC. Depending on the polynucleotide that hybridizes to a “portion” of the polynucleotide, at least about 15 nucleotides (nt) of the reference polynucleotide, and more preferably at least about 20 nt, even more preferably at least about 30 nt, and even more preferably about Polynucleotides (either DNA or RNA) that hybridize to polynucleotides longer than 30 nt are contemplated. Polynucleotides (oligonucleotides) that hybridize to “parts” of such polynucleotides are also useful as detection probes as discussed in more detail herein.

  As described above, the polynucleotide according to the present invention is a polynucleotide that encodes a polypeptide that increases the number of primary branches without increasing the ratio of the number of secondary branches to the number of primary branches in rice. (1) a polynucleotide comprising the base sequence shown in SEQ ID NO: 1 or 2; (2) one or several bases in the base sequence shown in SEQ ID NO: 1 or 2; A polynucleotide comprising a nucleotide sequence substituted, deleted or added; (3) complementary to a nucleotide sequence in which one or several bases are substituted, deleted or added in the nucleotide sequence shown in SEQ ID NO: 1 or 2; A polynucleotide that hybridizes with a polynucleotide comprising the sequence under stringent conditions; (4) represented by SEQ ID NO: 3 A polynucleotide encoding a polypeptide comprising a amino acid sequence; or (5) a polypeptide comprising an amino acid sequence in which one or several amino acids are substituted, deleted or added in the amino acid sequence represented by SEQ ID NO: 3 Encoding polynucleotide.

  The polynucleotide according to the present invention may contain a sequence such as a sequence of an untranslated region (UTR) or a vector sequence (including an expression vector sequence).

  The vector according to the present invention can be prepared by inserting the polynucleotide according to the present invention into a predetermined vector by a known gene recombination technique. The vector is not limited to this, but a cloning vector can be used in addition to the recombinant expression vector described below.

  Although it does not specifically limit as a supply source for acquiring the polynucleotide based on this invention, It is preferable that it is a biological material. As used herein, the term “biological material” is intended to be a biological sample (a tissue sample or cell sample obtained from an organism), and in the examples described below, rice is used. However, it is not limited to this.

[2: Polypeptide]
In one aspect, the present invention provides a polypeptide that increases the number of primary branches without increasing the ratio of the number of secondary branches to the number of primary branches in rice.

  The polypeptide according to the present invention is preferably a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3 or a variant thereof.

  As used herein with respect to proteins or polypeptides, the term “variant” intends a polypeptide that retains a specific activity of the polypeptide of interest, and consists of an amino acid sequence shown in SEQ ID NO: 2. By “polypeptide variant” is intended a polypeptide having the function of increasing the number of primary branches without increasing the ratio of the number of secondary branches to the number of primary branches in rice.

  It is well known in the art that some amino acids in the amino acid sequence that make up a polypeptide can be easily modified without significantly affecting the structure or function of the polypeptide. Furthermore, it is also well known that there are variants in natural proteins that do not significantly alter the structure or function of the protein, not only artificially modified.

  One skilled in the art can readily mutate one or several amino acids in the amino acid sequence of a polypeptide using well-known techniques. For example, according to a known point mutation introduction method, an arbitrary base of a polynucleotide encoding a polypeptide can be mutated. In addition, a deletion mutant or an addition mutant can be prepared by designing a primer corresponding to an arbitrary site of a polynucleotide encoding a polypeptide.

  The polypeptide according to the present invention, as shown in FIG. 10, is the 15th, 39th, 162nd, 195th, 226th, 289th, in the amino acid sequence shown in SEQ ID NO: 3. At the 295th, 366th, and 412th positions, amino acids are different from other rice. In addition, there is a deletion of 3 amino acids in the region from the 309th position to the 314th position in the amino acid sequence. Since these differences are thought to produce a function of increasing the number of primary branch infarcts without increasing the ratio of the number of secondary branch infarcts to the number of primary branch infarcts in rice, these amino acids are conserved. preferable. That is, the mutation of the polypeptide according to the present invention from the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3 is the first to the 14th, 16th to 38th of the amino acid sequence shown in SEQ ID NO: 3. , 40th to 161st, 163rd to 194th, 196th to 225th, 227th to 288th, 290th to 294th, 296th to 308th, It is preferable to occur at the 315th to 365th, the 367th to 411st, or the 413th to 425th. However, as long as the polypeptide has a function of increasing the number of primary branch infarcts without increasing the ratio of the number of secondary branch infarcts to the number of primary branch infarcts in rice, mutations may occur other than the above positions.

  As described above, the polypeptide according to the present embodiment is a polypeptide having an activity of increasing the number of primary branch infarcts without increasing the ratio of the number of secondary branch infarcts to the number of primary branch infarcts in rice, (1) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3 or (2) a polypeptide consisting of an amino acid sequence in which one or several amino acids are substituted, deleted or added in the amino acid sequence shown in SEQ ID NO: 3 Those skilled in the art will readily understand that such polypeptides can be encoded by the polynucleotides of the present invention.

  Polypeptides according to the present invention include natural purified products, products of chemical synthesis procedures, and prokaryotic or eukaryotic hosts (eg, bacterial cells, yeast cells, higher plant cells, insect cells, and mammalian cells). ) From recombinantly produced products. Depending on the host used in the recombinant production procedure, the polypeptides according to the invention may be glycosylated or non-glycosylated. Furthermore, the polypeptides according to the invention may also contain an initiating modified methionine residue in some cases as a result of host-mediated processes.

  The polypeptide according to the present invention may be a polypeptide in which amino acids are peptide-bonded, but is not limited thereto, and may be a complex polypeptide including a structure other than the polypeptide. As used herein, examples of the “structure other than the polypeptide” include sugar chains and isoprenoid groups, but are not particularly limited.

  The polypeptide according to the present invention may include an additional polypeptide. Examples of the additional polypeptide include epitope-tagged polypeptides such as His, Myc, and Flag.

  In another aspect, the present invention provides a method for producing a polypeptide that increases the number of primary branches without increasing the ratio of the number of secondary branches to the number of primary branches in rice.

  In one embodiment, the method for producing a polypeptide according to the present invention uses a vector containing a polynucleotide encoding the polypeptide.

  In one aspect of this embodiment, it is preferable that the vector is used in a recombinant expression system in the method for producing a polypeptide according to this embodiment. When a recombinant expression system is used, a polynucleotide obtained by incorporating a polynucleotide encoding the polypeptide of the present invention into a recombinant expression vector, introducing it into a host capable of expression by a known method, and translating it in the host. For example, a method of purifying a peptide can be employed. The recombinant expression vector may or may not be a plasmid, as long as the target polynucleotide can be introduced into the host. Preferably, the method for producing a polypeptide according to this embodiment includes a step of introducing the vector into a host.

  Thus, when introducing a foreign polynucleotide into a host, the expression vector preferably incorporates a promoter that functions in the host so as to express the foreign polynucleotide. The method for purifying a recombinantly produced polypeptide differs depending on the host used and the nature of the polypeptide, but the target polypeptide can be purified relatively easily by using a tag or the like.

  The method for producing a polypeptide according to this embodiment preferably further includes a step of purifying the polypeptide from an extract of cells or tissues containing the polypeptide. The step of purifying a polypeptide is to prepare a cell extract from cells or tissues by a well-known method (for example, a method in which a cell or tissue is disrupted and then centrifuged to collect a soluble fraction). Known methods (eg ammonium sulfate precipitation or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography) The step of purifying by a) is preferred, but not limited thereto. Most preferably, high performance liquid chromatography (“HPLC”) is used for purification.

  In another aspect of the present embodiment, the method for producing a polypeptide according to the present embodiment is preferably such that the vector is used in a cell-free protein synthesis system. When using a cell-free protein synthesis system, various commercially available kits can be used. Preferably, the method for producing a polypeptide according to this embodiment includes a step of incubating the vector and a cell-free protein synthesis solution.

  The cell-free protein synthesis system is a technique widely used for identification of various proteins encoded by intracellular mRNA and cloned cDNA. The cell-free protein synthesis system (cell-free protein synthesis method, cell-free protein translation system) The cell-free protein synthesis solution is also used.

  Cell-free protein synthesis systems include systems using wheat germ extract, systems using rabbit reticulocyte extract, systems using Escherichia coli S30 extract, and cell component extracts obtained from plant devolatilized protoplasts. . Generally, eukaryotic genes are selected for translation of eukaryotic genes, ie, systems using wheat germ extract or systems using rabbit reticulocyte extract. In view of the origin of the gene (prokaryotes / eukaryotes) and the intended use of the protein after synthesis, it may be selected from the above synthesis system.

  Many virus-derived gene products express their activity after translation through complex biochemical reactions involving intracellular membranes such as the endoplasmic reticulum and Golgi apparatus. In order to reproduce the above, it is necessary to add an intracellular membrane component (for example, a microsomal membrane). A cell component extract obtained from plant evacuated protoplasts is preferred because it can be used as a cell-free protein synthesis solution retaining an intracellular membrane component, and therefore does not require the addition of a microsomal membrane.

  As used herein, “intracellular membrane component” refers to an organelle composed of a lipid membrane present in the cytoplasm (ie, cells such as the endoplasmic reticulum, Golgi apparatus, mitochondria, chloroplast, vacuole and the like). Inner granules in general) are intended. In particular, the endoplasmic reticulum and the Golgi apparatus play an important role in post-translational modification of proteins, and are essential cellular components for the maturation of membrane proteins and secreted proteins.

  In another embodiment, the method for producing a polypeptide according to the present invention preferably purifies the polypeptide from cells or tissues that naturally express the polypeptide. The method for producing a polypeptide according to this embodiment preferably includes a step of identifying a cell or tissue that naturally expresses the polypeptide according to the present invention using an antibody or oligonucleotide described below. In addition, the polypeptide production method according to the present embodiment preferably further includes a step of purifying the polypeptide.

  In still another embodiment, the method for producing a polypeptide according to the present invention chemically synthesizes a polypeptide according to the present invention. Those skilled in the art will easily understand that the polypeptide according to the present invention can be chemically synthesized by applying a well-known chemical synthesis technique based on the amino acid sequence of the polypeptide according to the present invention described herein. .

  As described above, the polypeptide obtained by the method for producing a polypeptide according to the present invention may be a naturally occurring mutant polypeptide or an artificially prepared mutant polypeptide.

  Thus, it can be said that the method for producing a polypeptide according to the present invention may use a known and conventional technique based on at least the amino acid sequence of the polypeptide or the base sequence of the polynucleotide encoding the polypeptide. That is, it should be noted that a production method including steps other than the various steps described above also belongs to the technical scope of the present invention.

[3: Antibody]
The present invention provides an antibody that specifically binds to a polypeptide that increases the number of primary branches without increasing the ratio of the number of secondary branches to the number of primary branches in rice. The antibody according to the present invention is not limited as long as it can specifically bind to the polypeptide, and may be a polyclonal antibody against the polypeptide, but is preferably a monoclonal antibody against the polypeptide. Monoclonal antibodies have advantages such as uniform properties, easy supply, and semipermanent storage as hybridomas.

  As used herein, the term “antibody” means an immunoglobulin (IgA, IgD, IgE, IgG, IgM and their Fab fragments, F (ab ′) 2 fragments, Fc fragments), eg Examples include, but are not limited to, polyclonal antibodies, monoclonal antibodies, single chain antibodies, and anti-idiotype antibodies.

  “Antibodies” can be prepared according to various known methods. For example, monoclonal antibodies can be prepared by techniques known in the art (eg, hybridoma method (Kohler, G. and Milstein, C., Nature 256, 495-497 (1975)), trioma method, human B-cell hybridoma method (Kozbor). , Immunology Today 4, 72 (1983)) and the EBV-hybridoma method (see Monoclonal Antibodies and Cancer Therapy, Alan R Lis, Inc., 77-96 (1985)). .

  Peptide antibodies can also be obtained by methods known in the art (eg, Chow, M. et al., Proc. Natl. Acad. Sci. USA 82: 910-914; and Bittle, FJ et al., J. Gen. Virol. 66: 2347-2354 (1985), which is incorporated by reference herein, can be readily prepared.

  It will be apparent to those skilled in the art that Fab and F (ab ') 2 and other fragments of the antibodies according to the invention can be used in accordance with the methods disclosed herein. Such fragments can typically be produced by proteolytic cleavage using an enzyme such as papain (resulting in a Fab fragment) or pepsin (resulting in an F (ab ') 2 fragment). Alternatively, fragments that bind to a polypeptide according to the invention can be produced by the application of recombinant DNA technology or by chemical synthesis.

  As described above, the antibody according to the present embodiment only needs to have a fragment (for example, Fab fragment and F (ab ′) 2 fragment) that specifically binds to the polypeptide according to the present invention. It should be noted that immunoglobulins consisting of Fc fragments are also included in the present invention.

  That is, an object of the present invention is to provide an antibody that specifically binds to the polypeptide according to the present invention and use thereof, and the types of individual immunoglobulins specifically described in the present specification. (IgA, IgD, IgE, IgG, or IgM) does not exist in an antibody production method or the like. Therefore, it should be noted that antibodies obtained by methods other than the above methods also belong to the scope of the present invention.

[4: Production method and production kit of rice in which the number of primary branches is increased without increasing the ratio of the number of secondary branches to the number of primary branches]
The present invention provides a method for producing rice with an increased number of primary branches without increasing the ratio of the number of secondary branches to the number of primary branches. The rice production method according to the present invention only needs to include a step of introducing the polynucleotide according to the present invention into a plant body. In one embodiment, the method for producing rice according to the present invention includes a rice individual not expressing the polynucleotide according to the present invention (gene non-expressing body) and a rice individual expressing the polynucleotide according to the present invention ( The method includes a step of mating with a gene expression body), and is a preferable method for producing rice that is not a transformant. At this time, it is preferable to carry out backcrossing in order to preserve the traits of the non-gene-expressing body and avoid the influence of the gene-expressing body. Also, once a gene expression body is obtained by performing backcrossing and the background is completely a gene non-expressing gene, the number of primary branch infarcts can be easily determined by crossing the gene expression body with the gene non-expressing body. Rice having an increased number of primary branch infarcts can be produced without increasing the ratio of the number of secondary branch infarcts.

  In another embodiment, the method for producing a plant according to the present invention introduces a recombinant vector containing the polynucleotide according to the present invention into a plant so that the polypeptide encoded by the polynucleotide can be expressed. It is a method including a process.

  When a recombinant expression vector is used, the vector to be used for plant transformation is not particularly limited as long as it is a vector capable of expressing the polynucleotide according to the present invention in the plant. Examples of such a vector include a vector having a promoter that constitutively expresses a polynucleotide in a plant cell (eg, cauliflower mosaic virus 35S promoter), or a promoter that is inducibly activated by an external stimulus. The vector which has is mentioned.

  Plants to be transformed in the present invention include whole plants, plant organs (eg leaves, petals, stems, roots, seeds, etc.), plant tissues (eg epidermis, phloem, soft tissue, xylem, vascular bundle, It means any of a palisade tissue, a spongy tissue, etc.) or a plant culture cell, or various forms of plant cells (eg, suspension culture cells), protoplasts, leaf sections, callus, and the like. Although it does not specifically limit as a plant used for transformation, It is preferable that it is a rice, and it is more preferable that it is a rice of a japonica type, a Javanica type, or a tropical Javanica type.

  For the introduction of genes into plants, transformation methods known to those skilled in the art (for example, the Agrobacterium method, gene gun, PEG method, electroporation method, etc.) are used. It is divided roughly into the method of introducing into cells. When the Agrobacterium method is used, the constructed plant expression vector is introduced into an appropriate Agrobacterium (for example, Rhizobium radiobacter (formerly Agrobacterium tumefaciens)). The strain can be infected with a sterile leaf according to the leaf disk method (Hirofumi Uchimiya, Plant Gene Manipulation Manual (1990), pages 27-31, Kodansha Scientific, Tokyo) to obtain a transformed plant. Nagel et al. (Micibiol. Lett., 67, 325 (1990)), which can be used for example by first introducing an expression vector into Agrobacterium and then transformed into Agrobacterium. The thermium is a method of introducing plant cells or plant tissues by the method described in Plant Molecular Biology Manual (SB Gelvin et al., Academic Press Publishers), where “plant tissue” is obtained by culturing plant cells. When transformation is performed using the Agrobacterium method, pBI binary vectors (for example, pBIG, pBIN19, pBI101, pBI121, pBI221, and pPZP202) can be used.

  Electroporation and gene gun methods are known as methods for directly introducing genes into plant cells or plant tissues. When using a gene gun, the plant body, plant organ, plant tissue itself may be used as it is as a target for gene introduction, or it may be used after preparing a section, or a protoplast is prepared and used. Also good. The sample prepared in this manner can be processed using a gene transfer apparatus (for example, PDS-1000 (BIO-RAD)). The treatment conditions vary depending on the plant or sample, but are usually performed at a pressure of about 450 to 2000 psi and a distance of about 4 to 12 cm.

  A cell or plant tissue into which a gene has been introduced is first selected for drug resistance such as hygromycin resistance, and then regenerated into a plant by a conventional method. Regeneration of a plant body from a transformed cell can be performed by a method known to those skilled in the art depending on the type of plant cell. The selection marker is not limited to hygromycin resistance, and examples thereof include drug resistance to bleomycin, kanamycin, gentamicin, chloramphenicol and the like.

  When plant cultured cells are used as a host, the recombinant vector can be obtained by, for example, microinjection, electroporation (electroporation), polyethylene glycol, gene gun (particle gun), protoplast fusion, calcium phosphate, etc. It is introduced into a cultured cell and transformed. Callus, shoots, hairy roots, etc. obtained as a result of transformation can be used for cell culture, tissue culture or organ culture as they are, and can be used at a suitable concentration using a conventionally known plant tissue culture method. Of plant hormones (auxin, cytokinin, gibberellin, abscisic acid, ethylene, brassinolide, etc.).

  Whether or not a gene has been introduced into a plant can be confirmed by PCR, Southern hybridization, Northern hybridization, or the like. For example, DNA is prepared from a transformed plant, PCR is performed by designing a DNA-specific primer. PCR can be performed under the same conditions as those used for preparing the plasmid. Thereafter, the amplified product is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis or capillary electrophoresis, stained with ethidium bromide, SYBR Green solution, etc., and the amplified product is detected as a single band, It can be confirmed that it has been transformed. Moreover, PCR can be performed using a primer previously labeled with a fluorescent dye or the like to detect an amplification product. Furthermore, it is possible to employ a method in which the amplification product is bound to a solid phase such as a microplate and the amplification product is confirmed by fluorescence or enzymatic reaction.

  Once rice in which the polynucleotide of the present invention has been integrated into the genome is obtained, offspring can be obtained by sexual or asexual reproduction of the rice. In addition, for example, seeds, fruits, cuttings, tubers, tuberous roots, strains, callus, protoplasts, and the like can be obtained from the rice or its progeny, and the rice can be mass-produced based on these. Therefore, the present invention also includes a plant into which the polynucleotide according to the present invention is introduced so that it can be expressed, or a progeny of the plant having the same properties as the plant, or a tissue derived therefrom.

  The present invention further provides a kit for producing rice having an increased number of primary branches without increasing the ratio of the number of secondary branches to the number of primary branches. As used herein, a “kit” is intended to be a form in which at least one of various components is contained in another substance. In the present specification, a kit used for producing rice is referred to as a “creation kit”.

  The production kit according to the present invention may be provided with the polynucleotide according to the present invention. In the production kit according to the present invention, the polynucleotide according to the present invention may be provided in an isolated state, or may be provided in the form of a plant having the polynucleotide. By introducing the isolated polynucleotide of the present invention into rice, a rice plant having an increased number of primary branch infarcts without increasing the ratio of the number of secondary branch infarcts to the number of primary branch infarcts is produced. Can do. In addition, by mating rice having the polynucleotide according to the present invention and other rice, a rice plant having an increased number of primary branch infarcts without increasing the ratio of the number of secondary branch infarcts to the number of primary branch infarcts is produced. Can do. Therefore, those skilled in the art will understand that the production kit according to the present invention can be suitably used to produce rice with an increased number of primary branch infarcts without increasing the ratio of the number of secondary branch infarcts to the number of primary branch infarcts. Easy to understand.

[5: Rice Screening Method and Screening Kit with Increased Primary Branch Infarct Number without Increasing the Ratio of Secondary Branch Infarct Number to Primary Branch Infarct Number]
The present invention provides a method for screening rice having an increased number of primary branches without increasing the ratio of the number of secondary branches to the number of primary branches.

  In one embodiment, the screening method according to the present invention includes a step of incubating an oligonucleotide consisting of a fragment of the polynucleotide according to the present invention or a complementary sequence thereof with an extract from a plant (eg, rice). Preferably, the screening method according to the present invention includes a step of hybridizing the target plant-derived genomic DNA, mRNA or cDNA for mRNA with the oligonucleotide according to the present invention.

  By using the screening method according to the present invention, by detecting the target polynucleotide to be hybridized, rice having an increased number of primary branch infarcts can be easily obtained without increasing the ratio of the number of secondary branch infarcts to the number of primary branch infarcts. Can be detected (screened).

  As used herein, the term “oligonucleotide” is intended to be a combination of several to several tens or hundreds of nucleotides, and is used interchangeably with “polynucleotide”. Oligonucleotides are called dinucleotides (dimers) and trinucleotides (trimers) for short ones, and 30 mers (also called 30 bases and 30 nucleotides) or 100 mer (also called 100 bases and 100 nucleotides) for long ones. And the number of nucleotides polymerized. Oligonucleotides can be produced as fragments of longer polynucleotides or chemically synthesized.

  The oligonucleotide used in the screening method according to the present invention can be used as a PCR primer or a hybridization probe for obtaining the polynucleotide according to the present invention or a fragment thereof.

  In addition, those skilled in the art are attributed to the hybridization that occurs between the oligonucleotide used in the screening method according to the present invention and the target gene (polynucleotide) in any of the above-described applications. It is easily understood that it is used to hybridize with a gene of interest (polynucleotide).

  A fragment of a polynucleotide according to the present invention comprises at least 7 nucleotides (nt), 10 nt, 12 nt, preferably about 15 nt, and more preferably at least about 20 nt, even more preferably at least about 30 nt, and even more preferably at least about 40 nt. However, a person skilled in the art can appropriately set a preferable length according to the above-described use. By “a fragment having a length of at least 20 nt”, for example, a fragment comprising 20 or more consecutive base sequences from the base sequence shown in SEQ ID NO: 2 or a complementary sequence thereof is intended. By referring to the present specification, the base sequence shown in SEQ ID NO: 2 is provided, so that those skilled in the art can easily prepare a DNA fragment based on SEQ ID NO: 2. For example, restriction endonuclease cleavage or ultrasonic shearing can be readily used to create fragments of various sizes. Alternatively, such fragments can be made synthetically. Appropriate fragments (oligonucleotides) are synthesized by Applied Biosystems Incorporated (ABI, 850 Lincoln Center Dr., Foster City, CA 94404) type 392 synthesizer and the like.

  Oligonucleotides used in the screening method according to the present invention include not only double-stranded DNA but also single-stranded DNA and RNA such as sense strand and antisense strand constituting the same. The oligonucleotide can be used as a tool for gene expression manipulation by an antisense RNA mechanism. Antisense RNA technology reduces gene products derived from endogenous genes. By introducing the oligonucleotide, it is possible to reduce the content of the polypeptide having the activity to increase the number of primary branch infarct without increasing the ratio of the number of secondary branch to the number of primary branch infarction. The number of next branch infarcts can be controlled.

  Thus, the oligonucleotide used in the screening method according to the present invention detects a polynucleotide that encodes a polypeptide that increases the number of primary branches without increasing the ratio of the number of secondary branches to the number of primary branches. By using it as a hybridization probe or a primer for amplifying the polynucleotide, a plant or tissue expressing a polypeptide having the activity can be easily detected (screened). Furthermore, using the above-mentioned oligonucleotide as an antisense oligonucleotide, expression of a polypeptide that increases the number of primary branches without increasing the ratio of the number of secondary branches to the number of primary branches in the plant or its tissue or cell Can be controlled.

  In another embodiment, the method for screening rice with increased primary branch infarct without increasing the ratio of the number of secondary branch to the number of primary branch in accordance with the present invention is an extract from a plant (eg, rice). Is reacted with the antibody according to the present invention to detect the polypeptide according to the present invention. As described above, the antibody specifically binds to the polypeptide according to the present invention to form an immune complex, so that the primary branch expressed in the plant body is detected by detecting the formation of the complex. A polypeptide that increases the number of primary branch infarcts can be easily detected without increasing the ratio of the number of secondary branch infarcts to the number of infarcts. That is, according to the screening method of the present invention, rice having an increased number of primary branch infarcts can be screened without increasing the ratio of the number of secondary branch infarcts to the number of primary branch infarcts. Formation of the complex can be detected using, for example, a method in which the antibody is previously labeled with an isotope or a method using a secondary antibody against the antibody. Specifically, the screening method may be a well-known Western blot method, but is not limited thereto. Moreover, although the extract from the said plant can be produced using the freezing crushing method using liquid nitrogen, and a commercially available extraction kit, it is not limited to these. In addition, as used herein, the “extract” may be a crude product or a purified product that has undergone several purification steps.

  The present invention further provides a kit for screening rice having an increased number of primary branches without increasing the ratio of the number of secondary branches to the number of primary branches. In the present specification, a kit used for screening rice is referred to as a “screening kit”.

  In one embodiment, the screening kit according to the present invention comprises an oligonucleotide consisting of a fragment of the polynucleotide according to the present invention or a complementary sequence thereof. As described above, incubating the oligonucleotide with an extract from a plant (eg, rice) increased the number of primary branches without increasing the ratio of the number of secondary branches to the number of primary branches. Rice can be screened. Therefore, the screening kit according to the present invention can be suitably used for screening rice having an increased number of primary branches without increasing the ratio of the number of secondary branches to the number of primary branches. Is easy to understand.

  In another embodiment, the screening kit according to the present invention comprises the antibody according to the present invention. As described above, by reacting an extract from a plant (eg, rice) with the antibody according to the present invention, the number of primary branch infarcts increases without increasing the ratio of the number of secondary branch infarcts to the number of primary branch infarcts. The detected rice can be detected. Therefore, the screening kit according to the present invention can be suitably used for screening rice having an increased number of primary branch infarcts without increasing the ratio of the number of secondary branch infarcts to the number of primary branch infarcts. Is easy to understand.

  The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope shown in the claims, and embodiments obtained by appropriately combining technical means disclosed in different embodiments. Is also included in the technical scope of the present invention.

  Moreover, all the academic literatures and patent literatures described in this specification are incorporated herein by reference.

[Example 1: QTL analysis]
Sasanishiki as a parent, Habataki was crossed, and Sasanishiki was backcrossed continuously from the National Institute of Agrobiological Sciences. Sasanishiki is a japonica-type rice variety, and Habataki is an indica-type rice variety. In addition, it is known that the number of primary branch infarcts in Habataki and the ratio of the number of secondary branch infarcts to the number of primary branch infarcts is larger than that of Sasanishiki.

  QTL analysis was performed on the series obtained from the above continuous backcross. As a result, QTL was found on chromosome 6 that the gene derived from Habataki increases the number of primary branches without increasing the ratio of the number of secondary branches to the number of primary branches (FIG. 2).

[Example 2: Isolation of causative gene]
In order to isolate the QTL causative gene obtained in Example 1, in general, the phenotype of rice having a candidate gene of the causative gene is observed. At that time, it is known that quantitative traits such as the number of primary and secondary branch infestations vary depending on environmental factors, so that rice grown in the same environment as the actual farming environment of general farmers It is preferred to observe the phenotype. For this reason, rice having a candidate gene for the above causative gene is not a transformant but is usually obtained by mating. Whether or not the rice obtained by mating contains the candidate gene has been left to chance, so it was thought that it was very difficult to isolate the causative gene of the QTL. Et al. Isolated the causative gene by the following procedure.

  First, from the results of the QTL analysis in Example 1, it was estimated that the region where the above-mentioned QTL causal gene may exist is a region sandwiched between known markers G329 and R2549EH and its peripheral region (FIG. 2).

  Next, in the estimated region, a large number of lines were obtained by self-breeding rice lines having heterogeneous genes derived from Sasanishiki and Habataki. Usually, about 2000 to 3000 lines are used for isolation of causative genes from the results of QTL analysis, but the present inventors have created about 10,000 lines based on their own findings. By detecting the DNA marker for each individual, it was detected at which position the transfer occurred in each individual and which region was the gene derived from Habataki or Sasanishiki. In addition to known markers published in the Rice Genome Project (http://rgp.dna.affrc.go.jp/), DNA markers uniquely designed to detect a narrower range of crossovers Was used. Unique markers include markers designed to include restriction enzyme sites or repeat sequences based on the sequence of Nipponbare (a japonica-type rice variety) published in the Rice Genome Project, and sequences based on Sasanishiki and Habataki. The designed marker was used.

  Seeds that were self-pollinated offspring of each individual were harvested and sown in the following year. The phenotype of the individual was confirmed by comparing the phenotype of the next year. That is, the number of primary branch infarcts in the following fiscal year is increased (hereinafter referred to as Habataki type) or not increased (hereinafter referred to as Sabataki type), or Habataki type and Sasanishiki type. Is present (hereinafter referred to as a separation type).

  The results are shown in FIG. Symbols beginning with M- indicate markers uniquely designed by the inventors. A, B, and H mean that the corresponding marker sites are Sasanishiki-derived homotype, Habataki-derived homotype, and Sasanishiki-derived and Habataki-derived heterotype, respectively. A portion where A, B, or H is overlapped indicates the result of performing a plurality of times. As shown in FIG. 3, if the region sandwiched between the markers M-7 and M-56 (the region surrounded by the square in the figure) is derived from Habataki, the phenotype also became Habataki. FIG. 4, Table 1 and Table 2 show a detailed comparison of the respective trait values of rice from which the region is derived from Habataki, rice derived from Sasanishiki, and rice which is heterogeneous from Sasanishiki and Habataki.

FIG. 4 is a graph comparing the number of primary branch infarcts in rice where the region is derived from Habataki and rice derived from Sasanishiki. As shown, the number of primary branch infarcts is higher in rice where the region is derived from Habataki than in rice where the region is derived from Sasanishiki.

  Table 1 and Table 2 show the respective trait values of rice from which the region is derived from Habataki, rice derived from Sasanishiki, and heterozygous rice derived from Sasanishiki and Habataki. Tables 1 and 2 show the results of field tests in 2004 and 2005, respectively. “Primary” indicates the number of primary branch infarctions, “Secondary” indicates the number of secondary branch infarctions, and “Secondary / primary” indicates the ratio of the number of secondary branch infarctions to the number of primary branch infarctions. “HI” indicates a harvest index. The harvest index is a value obtained by dividing the total weight of the cocoon by the weight of the whole plant. “Number of sinking dredging” indicates the number of dredging sinks in the water. “Grain weight” indicates the weight (g) per thousand grains. “Subsidence weight” indicates the total weight of the dredged sun. “Sterility ratio” indicates the ratio of cocoons containing no rice. As shown in Tables 1 and 2, in the rice in which the region is derived from Habataki, the ratio of the primary branch infarct number to the primary branch infarction is not increased compared to rice in which the region is derived from Sasanishiki. The number of infarcts increased and the harvest index improved. In addition, the sterility ratio, which is one of the indices indicating the degree of ripening, hardly changed.

  The region sandwiched between the markers M-7 and M-56 includes a region that is clearly presumed to be a gene (hereinafter, the gene name is referred to as OsPRB1) and a region that may be another gene (hereinafter, gene name). Was referred to as OsHI1). Therefore, we further analyzed rice lines in which transfer between the two genes occurred. In addition, although the rice strains as described above cannot usually be obtained, as described above, the present inventors have created about 10,000 strains based on their own knowledge. I was able to.

  FIG. 5 shows the genotype of a strain in which the region containing OsPRB1 is derived from Habataki and the region containing OsHI1 is derived from Sasanishiki. In FIG. 6, regions containing OsPRB1 and not containing OsHI1 (regions sandwiched between marker M-41 and marker M-56) are rice derived from Habataki, rice derived from Sasanishiki, and heterozygous derived from Sasanishiki and Habataki The distribution of the number of primary branch infarcts in a certain rice is shown. As shown in FIG. 6, if OsPRB1 was derived from Habataki, the number of primary branch infarcts was increased even if OsHI1 was derived from Sasanishiki. Table 3 and Table 4 show the respective trait values of rice derived from Habataki, rice derived from Sasanishiki, and heterozygous rice derived from Sasanishiki and Habataki. Tables 3 and 4 show the results of the pot test and the field test in the growth chamber in 2006, respectively.

As shown in Tables 3 and 4, if OsPRB1 is derived from Habataki, even if OsHI1 is derived from Sasanishiki, the ratio of the number of primary branch infarcts to the number of primary branch infarcts does not increase. It was increasing. In addition, the proportion of sterility was almost unchanged. On the other hand, there was no improvement in the harvest index.

  FIG. 7 shows the genotype of a strain in which the region containing OsPRB1 is derived from Sasanishiki and the region containing OsHI1 is derived from Habataki. Tables 5 and 6 show that the region containing OsHI1 but not OsPRB1 (the region sandwiched between the marker M-7 and the marker M-55) is derived from Habataki, Rice derived from Sasanishiki, and Sasanishiki derived and Habataki. Each trait value of rice which is hetero from the origin is shown. Tables 3 and 4 show the results of the pot test and the field test in the growth chamber in 2006, respectively.

As shown in Tables 5 and 6, when OsPRB1 was derived from Sasanishiki, even when OsHI1 was derived from Habataki, the primary branch was not increased. On the other hand, the harvest index was improved, but the degree of improvement was small compared to the case where both OsPRB1 and OsHI1 were derived from Habataki.

  A summary of the above results is shown in FIGS. OsPRB1 and OsHI1 were present between about 64000th to about 74000th in the base sequence of AP003628 which is a PAC clone of chromosome 6 (FIG. 8). A rice line (05SHA422-12-8-18.31-b) in which the region containing OsPRB1 and OsHI1 is derived from Habataki has an increased number of primary branch infarcts without increasing the ratio of the number of secondary branch infarcts to the number of primary branch infarcts. It was increasing. Similarly, the rice line (05SHA 4221-29-7.8-b) in which only the region containing OsPRB1 is derived from Habataki increases the number of primary branch infarcts without increasing the ratio of the number of secondary branch infarcts to the number of primary branch infarcts. Was. On the other hand, in the rice line (05SHA4221-17-6.10-b) in which only the region containing OsHI1 is derived from Habataki, the number of primary branch infarctions did not increase (FIG. 9). From the above results, it was found that the causative gene of QTL causing the primary branch infarct number without increasing the ratio of the secondary branch infarct number to the primary branch infarct number is OsPRB1.

[Example 3: Sequencing]
The nucleotide sequences of Habataki, Sasanishiki, 05SHA422-12-8-18.31-b, and 05SHA4221-29-7.8-b were sequenced. As a result, the ORF of OsPRB1 and the position where the transfer occurred were detected.

  SEQ ID NO: 1 shows the nucleotide sequence from the position where the 05SHA 4221-29-7.8-b transfer occurred to the end of the ORF of Habataki OsPRB1. In addition, SEQ ID NO: 2 shows the nucleotide sequence from the position where the 05SHA422-12-8-18.31-b transfer occurred to the end of the ORF of Habataki OsPRB1. The ORF of OsPRB1 starts from the 2296th position of the base sequence shown in SEQ ID NO: 1 and ends at the 3658th position. FIG. 10 shows a comparison of the predicted amino acid sequences of OsPRB1 for Habataki, Sasanishiki, and Nipponbare. The large square on the upper right in FIG. 10 indicates a portion corresponding to F-BOX.

  APO1 has been reported as a gene having high homology to OsPRB1 (Patent Document 2). Patent Document 2 describes that the mutation of APO1 reduces the number of primary branch infarcts and the ratio of the number of secondary branch infarcts to the number of primary branch infarcts. That is, the mutation of APO1 described in Patent Document 2 does not increase the number of primary branches without increasing the ratio of the number of secondary branches to the number of primary branches, but even increases the number of primary branches. Absent. Therefore, those skilled in the art cannot easily come up with using the mutation of APO1 described in Patent Document 2 in order to breed high-yield rice. Furthermore, it is understood by those skilled in the art that rice having Habataki-derived OsPRB1 increases the number of primary branch infarcts without increasing the ratio of the number of secondary branch infarcts to the number of primary branch infarcts compared to rice without Hasaki-derived OsPRB1. Is unpredictable.

  The ORF of OsHI1 starts from the 3923rd position of the base sequence shown in SEQ ID NO: 2 and ends at the 2686th position.

[Example 4: Inactivation by RNAi]
Based on the Habataki OsPRB1 ORF, a vector for RNAi was prepared. Specifically, it consists of a base sequence (base sequence shown in SEQ ID NO: 4) in which the 2657th base is changed from C to T in the 2351st to 2724th bases of the base sequence shown in SEQ ID NO: 1. The polynucleotide was incorporated into a vector having a promoter sequence for RNAi. The base modification described above was performed in order to use a restriction enzyme (SacII) when incorporating the polynucleotide into a vector. Those skilled in the art will readily understand that modification of about one base can sufficiently function as RNAi. Moreover, those skilled in the art can easily understand that the nucleotide sequence of the polynucleotide is different from Sasanishiki OsPRB1 by only one base, and can act as RNAi against Sasanishiki OsPRB1. In Sasanishiki, the vector was introduced into Sasanishiki individuals, and the number of primary branch infarcts in the first breeding (T1) generation was observed. In the T1 generation, the vector is separated and dropped in some individuals.

  The results are shown in FIG. As shown, the number of primary branch infarcts decreased in the individual containing the vector compared to the individual not containing the vector. Therefore, it is considered that the polypeptide encoded by OsPRB1 has an effect of increasing the number of rice primary branch infarcts.

[Example 5: Transformation]
An expression vector containing Habataki OsPRB1 was designed. Specifically, a polynucleotide consisting of a sequence (base sequence shown in SEQ ID NO: 5) from upstream 1730 bp to downstream 790 bp of Habataki OsPRB1 was incorporated into an expression vector (pTN1, National Institute of Agrobiological Sciences). .

  The above vector was introduced into Sasanishiki individuals using a conventional method. For the T1 generation of the strain into which one copy of the vector was introduced, the number of primary branch infarcts of the individual containing the vector and the individual not containing the vector was compared (FIG. 12). As shown in FIG. 12, the number of primary branch infarcts increased in the individual containing the vector. Similarly, the relationship between the number of copies of the vector contained in the individual and the number of primary branch infarcts was investigated for the T1 generation of the strain into which two copies of the vector were introduced (FIG. 13). As shown in FIG. 13, the number of primary branch infarcts increased in individuals containing the vector compared to individuals not containing the vector at all, but there was no particular difference due to the copy number.

  By using the present invention, it is possible to increase the number of primary branch infarcts without increasing the ratio of the number of secondary branch infarcts to the number of rice primary branches. Can do.

It is explanatory drawing which shows the branch of rice. It is a graph which shows the result of QTL. It is a figure which shows the relationship between the genotype and phenotype of the system | strain which concerns on one Embodiment of this invention. It is a graph comparing the number of primary branch infarcts of rice having a polynucleotide according to the present invention with the number of primary branch infarcts of rice not having the polynucleotide. It is a figure which shows the genotype of the rice which concerns on one Embodiment of this invention. It is a graph which compares the number of primary branch infarction of rice which has the polynucleotide which concerns on this invention in a homo type or a hetero type, and the rice which does not have this polynucleotide. It is a figure which shows the genotype of rice. It is a schematic diagram which shows the position on the chromosome of the gene which concerns on this invention. It is a schematic diagram which shows the genotype of the rice which concerns on one Embodiment of this invention. It is a figure which compares the amino acid sequence of polypeptide which concerns on this invention, and its ortholog. It is a graph which compares the primary branch infarction number of the rice which inhibited the expression of the polypeptide which concerns on this invention using RNAi, and the primary branch infarction number of normal rice. It is a graph which compares the number of primary branch infarcts of rice transformed with the polynucleotide according to the present invention and the number of primary branch infarcts of rice not transformed. It is a graph which compares the number of primary branch infarcts of rice transformed with the polynucleotide according to the present invention and the number of primary branch infarcts of rice not transformed.

Claims (8)

A polynucleotide encoding a polypeptide comprising the amino acid sequence of any one of (A) and (B) below, and the number of primary branch infarcts without increasing the ratio of the number of secondary branch infarcts in rice A polynucleotide characterized in that it encodes a polypeptide capable of increasing
Amino acid sequence shown in (A) SEQ ID NO: 3; in the amino acid sequence shown in or (B) SEQ ID NO: 3, a single or several amino acids are substituted, added or deleted amino acid sequence, SEQ ID NO: 15th, 39th, 162nd, 195th, 226th, 289th, 295th, 366th and 412th amino acids in the amino acid sequence shown in Fig. 3 are conserved, An amino acid sequence to which no amino acid is added between or adjacent to the 309th to 314th positions . Following (A) ~ (C) of any one of the Turkey such the nucleotide sequence of claim 1, wherein the polynucleotide:
(A) the base sequence shown in SEQ ID NO: 1;
(B) a base sequence in which one or several nucleotides are substituted, added or deleted in the base sequence shown in SEQ ID NO: 1; or (C) a stringent sequence with a complementary sequence of the base sequence shown in SEQ ID NO: 1. A nucleotide sequence that can hybridize under conditions. The polynucleotide according to claim 1, which comprises any one of the following base sequences (A) to (C) and improves the harvest index in rice :
(A) the base sequence shown in SEQ ID NO: 2;
(B) 1 one or several nucleotides in the nucleotide sequence shown in SEQ ID NO: 2 is substituted, added or deleted the base sequence; or (C) the complementary sequence under stringent nucleotide sequence shown in SEQ ID NO: 2 hybridized obtained that base sequence in such conditions.   A polypeptide encoded by the polynucleotide according to claim 1 or 2.   A vector comprising the polynucleotide according to any one of claims 1 to 3.   A step of introducing the polynucleotide according to any one of claims 1 to 3 into rice, wherein the number of primary branch infarcts is increased without increasing the ratio of the number of secondary branch infarcts to the number of primary branch infarcts. Increased rice production method. Rice having an increased number of primary branch infarcts without increasing the ratio of the number of secondary branch infarcts to the number of primary branch infarcts, which is a descendant of rice produced by the method according to claim 6 .   Production of rice having an increased number of primary branch infarcts without increasing the ratio of the number of secondary branch infarcts to the number of primary branch infarcts, comprising the polynucleotide according to any one of claims 1 to 3. kit.