WO2009145290A1 - Plant having increased grain size which contains sh4 gene - Google Patents
Plant having increased grain size which contains sh4 gene Download PDFInfo
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- WO2009145290A1 WO2009145290A1 PCT/JP2009/059849 JP2009059849W WO2009145290A1 WO 2009145290 A1 WO2009145290 A1 WO 2009145290A1 JP 2009059849 W JP2009059849 W JP 2009059849W WO 2009145290 A1 WO2009145290 A1 WO 2009145290A1
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- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
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Definitions
- the present invention relates to a transformed plant introduced so that the sh4 gene is expressed. Further, the present invention relates to a plant body that has been cross-introduced so that a functional sh4 gene is expressed. The present invention also relates to a method for increasing the grain size of a plant body, which comprises the step of introducing a sh4 gene into the plant body.
- the present invention has been made in view of such a situation, and the problem is that a plant body can be obtained by introducing a sh4 gene into a plant body and expressing a functional sh4 gene discovered from wild rice.
- An object of the present invention is to provide a plant having an increased grain size.
- the present inventors have conducted intensive studies on genes involved in plant cultivation.
- the present inventors introduced a functional allele sh4 gene derived from wild rice into the cultivated rice by transformation, thereby increasing the culm as a new function and promoting translocation, resulting in a large grain size.
- T1 progenies T0-3 progeny-1, T0-3 progeny-2, T0-3 progeny-3, T0-5 progeny-1) of the line that introduced the sh4 gene into the breed Nipponbare were artificially Rice plants are cultivated in the meteorological chamber, fir weight per milligram (mg) (Fig. 7), total number of pods per individual (fruit seeds, sterile grains) (Fig. 8), and ears per individual The weight ( Figure 9) was measured. As a result, it was clarified that these individuals had significantly increased fir weight and ear weight (yield) for each individual compared to vector control and Nipponbare (FIGS. 7 to 9).
- the present invention provides the following (1) to (13).
- a plant having an increased grain size of the plant comprising the DNA according to any one of (a) to (d) below.
- D DNA that hybridizes under stringent conditions with DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1 or 2
- a transformed plant comprising the plant cell according to (6).
- a transformed plant that is a descendant or clone of the transformed plant according to (7).
- a method for increasing the grain size of a plant comprising the step of expressing the DNA according to any one of (a) to (d) below in a cell of the plant.
- T0-3 individuals From left to right, T0-3 individuals, self-breeding T1 progeny 3 individuals, T0-5 progeny 1 individual, vector control progeny 3 individuals, and Nipponbare 3 individuals are shown. It is a figure which shows the fir weight (mg) for every individual of the self-propagating T1 progeny individual of the system
- An object of the present invention is to provide a plant in which the grain size of the plant body is increased by introducing the functional sh4 gene into the plant body or expressing the functional sh4 gene discovered from wild rice. .
- the sh4 gene of the present invention is a gene in which all cultivated rice is deficient in function, according to multiple paper publications. It is considered that cultivated rice was established by the selection of a function-deficient type by ancient humans with the aim of reducing threshing properties in order to make it easier to cultivate in the initial process of rice cultivation. It is considered that there is no functional allele. This time, the functional allele sh4 gene has the effect of increasing the grain size of the plant body, so by transforming the plant with DNA encoding the protein, It is possible to grow plants with increased From simple considerations, a similar effect can be expected even in a near-isogenic replacement line in which a functional allele sh4 gene is introduced from wild rice by crossing.
- the plant into which the sh4 gene is introduced is not particularly limited, but is preferably a monocotyledonous plant, more preferably a gramineous plant, and most preferably cultivated rice.
- the varieties of gramineous plants are not particularly limited, but preferred examples include “Nipponbare”, “Nikomaru”, “Ochikara (great power)” and the like.
- “to increase the grain size of a plant” means to increase the volume and weight of the grain at the time of harvest by expressing the sh4 gene of the present invention in the plant. Moreover, the effect of increasing the size of the cocoon and the effect of promoting commutation also correspond to “increasing the size of the kernel of the plant”.
- the effect of increasing the size of the grain may be an effect that appears only in the process of generating the kernel of the plant. Moreover, the increase effect may be seen in all the grains, or the increase effect may be seen only in a specific grain.
- “whether the grain size of the plant body has increased” can be confirmed by measuring the fir weight (mg) per ear or the ear weight (g) per individual.
- the nucleotide sequence of the genomic DNA of the functional sh4 gene used in the present invention is SEQ ID NO: 1
- the nucleotide sequence of the ORF region of the gene is SEQ ID NO: 2
- the amino acid sequence of the protein encoded by the DNA is SEQ ID NO: : Shown in 3.
- the amino acid sequence of the function-deficient sh4 protein is shown in SEQ ID NO: 4.
- the DNA used in the present invention includes genomic DNA, genomic DNA, cDNA, and chemically synthesized DNA in chromosome fragments transferred by mating. Preparation of genomic DNA and cDNA can be performed by those skilled in the art using conventional means.
- genomic DNA for example, genomic DNA is extracted from rice varieties having the sh4 gene of the present invention, and a genomic library (plasmid, phage, cosmid, BAC, PAC, etc. can be used as a vector) It can be prepared by developing and performing colony hybridization or plaque hybridization using a probe prepared based on the DNA encoding the sh4 protein of the present invention (for example, SEQ ID NO: 2).
- a primer specific for the DNA encoding the sh4 protein of the present invention for example, SEQ ID NO: 2
- cDNA is synthesized based on mRNA extracted from rice varieties having the sh4 gene of the present invention, and inserted into a vector such as ⁇ ZAP to create a cDNA library. It can be prepared by performing colony hybridization or plaque hybridization in the same manner as described above, or by performing PCR.
- the DNA used in the present invention includes DNA encoding a protein functionally equivalent to the functional sh4 protein described in SEQ ID NO: 3.
- “having a function equivalent to the sh4 protein” means that the target protein has a function of increasing the grain size of the plant body.
- Such DNA is preferably derived from monocotyledonous plants, more preferably from gramineous plants, and most preferably from current wild rice.
- Such DNA includes, for example, mutants, derivatives, and the like that encode proteins consisting of amino acid sequences in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence set forth in SEQ ID NO: 3. Alleles, variants and homologs are included.
- the amino acid sequence of the encoded protein may be mutated in nature due to the mutation of the base sequence.
- the natural functional sh4 protein sequence
- it is included in the DNA of the present invention.
- the base sequence is mutated, it may not be accompanied by amino acid mutation in the protein (degenerate mutation), and such a degenerate mutant is also included in the DNA of the present invention.
- Whether or not a certain DNA encodes a protein having a function of increasing the grain size of a plant body can be evaluated as follows.
- the most general method is a method for examining the grain size of the plant body into which the DNA has been introduced. When the grain size of the plant body is increased, it can be seen that the introduced DNA encodes a protein having a function of increasing the grain size of the plant body.
- a hybridization reaction is preferably performed under stringent conditions.
- stringent hybridization conditions refer to conditions of 6M urea, 0.4% SDS, 0.5 ⁇ SSC, or equivalent stringency hybridization conditions. Isolation of DNA with higher homology can be expected by using conditions with higher stringency, for example, conditions of 6M urea, 0.4% SDS, 0.1 ⁇ SSC.
- the isolated DNA is considered to have high homology with the amino acid sequence of the sh4 protein (SEQ ID NO: 3) at the amino acid level.
- High homology means a sequence of at least 50% or more, more preferably 70% or more, more preferably 90% or more (for example, 95%, 96%, 97%, 98%, 99% or more) in the entire amino acid sequence.
- BLAST Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990, Proc Natl Acad Sci USA 90: 5873, 1993
- Programs called BLASTN and BLASTX based on the BLAST algorithm have been developed (Altschul SF, et al: J Mol Biol 215: 403, 1990).
- the present invention selects individuals having a functional allele of wild rice from the mating progeny of wild rice and cultivated rice, and a plant body whose grain size is larger than the parent, progeny, fixed line, variety, etc. Can provide.
- the introduction of the wild allele gene can confirm the patentability by satisfying both the increase in grain size and having the wild rice sh4 allele. Whether or not it has wild rice alleles can be confirmed by amplifying a specific genomic site by PCR, etc., and confirming the sequence of the DNA sequence using existing technology Can be confirmed.
- a transformed plant body in which the grain size of the plant body is increased using the DNA of the present invention can also be provided.
- DNA which may contain a control region
- the sh4 gene isolated by the present inventors has an effect of increasing the grain size of the plant body, but by introducing this sh4 gene into an arbitrary variety and overexpressing it, the grains of those lines It is possible to increase the size of the grains.
- the period required for this transformation is extremely short as compared with conventional gene transfer by crossing, and is advantageous in that it does not involve any other changes in traits.
- the present invention also provides a vector into which the DNA of the present invention is inserted.
- the vector of the present invention include a vector for expressing the DNA of the present invention in a plant cell for producing a transformed plant body.
- a vector is not particularly limited as long as it contains a promoter sequence that can be transcribed in plant cells and a terminator sequence including a polyadenylation site necessary for the stabilization of the transcript.
- the vector used for the transformation of plant cells is not particularly limited as long as the inserted gene can be expressed in the cells.
- plant cell includes various forms of plant cells, such as suspension culture cells, protoplasts, leaf sections, and callus.
- the vector of the present invention may contain a promoter for constitutively or inducibly expressing the protein of the present invention as well as the promoter inherent to the sh4 gene.
- promoters for constant expression include cauliflower mosaic virus 35S promoter, rice actin promoter, maize ubiquitin promoter, and the like.
- promoters for inducible expression are known to be expressed by external factors such as infection and invasion of filamentous fungi, bacteria, and viruses, low temperature, high temperature, drying, ultraviolet irradiation, and spraying of specific compounds. Promoters and the like.
- promoters include, for example, rice chitinase gene promoters expressed by infection and invasion of filamentous fungi, bacteria and viruses, tobacco PR protein gene promoters, rice lip19 gene promoters induced by low temperature, Rice "hsp80" and “hsp72” gene promoters induced by high temperature, Arabidopsis thaliana "rab16” gene promoter induced by drying, Parsley chalcone synthase gene promoter induced by UV irradiation, Anaerobic And the promoter of corn alcohol dehydrogenase gene induced under a certain condition.
- the rice chitinase gene promoter and tobacco PR protein gene promoter are also induced by specific compounds such as salicylic acid, and “rab16” is also induced by spraying the plant hormone abscisic acid.
- the present invention also provides a transformed cell into which the vector of the present invention has been introduced.
- the cells into which the vector of the present invention is introduced include plant cells for producing transformed plants. There is no restriction
- 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.
- various methods known to those skilled in the art such as polyethylene glycol method, electroporation (electroporation), Agrobacterium-mediated method, and particle gun method can be used.
- Regeneration of plant bodies from transformed plant cells can be performed by methods known to those skilled in the art depending on the type of plant cells.
- methods for producing transformed plants include gene transfer into protoplasts using polyethylene glycol and regeneration of plants (suitable for Indian rice varieties), gene transfer into protoplasts using electric pulses
- the method of regenerating plants Japanese rice varieties are suitable
- the method of directly introducing genes into cells by the particle gun method the method of regenerating plants, and the introduction of genes via Agrobacterium
- Several techniques, such as a method for regenerating plant bodies 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.
- the transformed plant cell can regenerate the plant body by redifferentiation.
- the method of redifferentiation varies depending on the type of plant cell. For example, for rice, the method of Fujimura et al. (Plant Tissue Culture Lett. 2:74 (1995)) can be mentioned, and for maize, Shillito et al. (Bio / Technology 7: 581 (1989)) and Gorden-Kamm et al. (Plant Cell 2: 603 (1990)). For potatoes, Visser et al. (Theor. Appl.
- the present invention includes a plant cell into which the DNA of the present invention has been introduced, a plant containing the cell, progeny and clones of the plant, and propagation material of the plant, its progeny and clones.
- Genomic DNA was extracted from wild rice Oryza nivara with A genome, and BAC library was created.
- BAC clones with sh4 gene region genomic fragments were designed with specific primers to increase only the sh4 region by PCR, and BAC clones with sh4 gene region were isolated with or without amplification by PCR, Then, after subcloning the DNA obtained by fragmenting the BAC clone DNA to a few kbp into the pUC18 vector, the end reading DNA sequence of each subclone was determined and assembled, so that the sh4 gene genomic region of O.nivara The DNA sequence was determined.
- the promoter region is predicted from the information of the known sh4 gene product, and the sh4 gene region, about 8.8 kbp length is excised by digestion reaction with KpnI and BamHI restriction enzyme, pPZP2H
- the genomic fragment was introduced into the -lac vector to create a transformation construct.
- an approximately 8.8 kb genomic fragment (FIG. 1, SEQ ID NO: 1) containing the coding region and the regulatory region of the isolated functional allele sh4 gene was transformed into two rice lines, Nipponbare, NIL (qSH1) (Konishi et al In 2006, the gene was introduced using the ultra-rapid transformation method of monocotyledons (Japanese Patent No. 314084) by the rice transformation method.
- Antibiotic hygromycin was used for selection of transformation. About 10 independent transformants were prepared, and various traits such as weight and shedding were measured.
- Example 2 Ripe rice seeds were harvested, and 5 seed grains were selected from each transformed line for each individual, and the mass was measured. Although there was a range of phenotypic changes presumed to be due to the position effect, a significant increase in weight was confirmed when compared with the vector control, which increased about 1.5 times depending on the line (FIG. 2). In terms of appearance, an increase in the size of the koji and an increase in the size of the brown rice were confirmed (FIG. 3). In addition, the effect on the number of spikelets was the same as that of the vector control, and almost no change was observed in the sh4 line (FIG. 4).
- Example 3 In the line in which the functional type sh4 was introduced, the ears attached to the T1 line of progeny progeny of individuals whose brown rice size was significantly increased were observed. An increase in the cocoon size equivalent to T0 was confirmed (FIG. 5).
- Example 4 According to the method described in Example 1, rice plants were cultivated in an artificial weather chamber for four self-breeding T1 progenies of the line that introduced the sh4 gene into the variety Nipponbare, and the fir weight (mg) per ear was measured. did. As a result, it was revealed that the fir weight was significantly increased as compared with vector control (a line in which only a vector was introduced into Nipponbare) and Nipponbare (FIG. 7). In addition, for these individuals, the total number of pods (fruit seeds, sterile grains) (FIG. 8) and panicle weight (FIG. 9) for each individual were measured and compared with Vector Control and Nipponbare.
- Example 5 In the same manner as in Example 1, the sh4 gene was introduced into the cultivar “Nikomaru”, which has the characteristics of good commutation, and the cultivar “Ochikara (large power)”, which has the characteristics that the grain of rice is large. Then, the average fir weight (mg) of the persimmon grains per transgenic T0 individual of these varieties was measured. As a result, it was confirmed that by introducing the sh4 gene in “Nikomaru”, rice grains become heavier as in Nipponbare (FIG. 10). In addition, it was clarified that the Ochikara was originally a large grain and the weight of one grain was 30 mg. However, by introducing the sh4 gene, a transformed line exceeding 40 mg was obtained (FIG. 11).
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Abstract
Disclosed is a plant having an increased grain size, which contains functional-type sh4 gene. Extensive studies have been made on genes involved in the grain sizes of plants, and it is found that a novel function of increasing the sizes of rice hulls, promoting translocation and, consequently, increasing the sizes of grains can be expressed in a rice plant cultivar ("Nihonbare", "Nikomaru", "Oochikara") by introducing a functional allele of sh4 gene derived from a wild-type rice plant into the rice plant cultivar by transformation.
Description
本発明は、sh4遺伝子が発現するように導入された形質転換植物体に関する。また、機能型sh4遺伝子が発現するように交配導入された植物体に関する。また、sh4遺伝子を植物体に導入する工程を含む、植物体の穀粒サイズを増大させる方法に関する。
The present invention relates to a transformed plant introduced so that the sh4 gene is expressed. Further, the present invention relates to a plant body that has been cross-introduced so that a functional sh4 gene is expressed. The present invention also relates to a method for increasing the grain size of a plant body, which comprises the step of introducing a sh4 gene into the plant body.
これまで、植物体の穀粒サイズに関していくつかの遺伝子が同定されているが、穀粒サイズを既知の一つの遺伝子の有無で改変することはこれまで容易ではなかった。
Li ら(非特許文献１)や Linら（非特許文献２）の報告によると、すべての栽培イネはsh4遺伝子に欠損を持つことで、脱粒性が比較的難になり、栽培化が進んだとされている。sh4遺伝子は既に単離され、報告されているが、その生物学的機能に関する知見は、脱粒性に関するもののみであった。 So far, several genes have been identified for the grain size of plants, but it has not been easy to modify the grain size with or without one known gene.
According to reports of Li et al. (Non-patent Document 1) and Lin et al. (Non-patent Document 2), all cultivated rice has a deficiency in the sh4 gene, which makes threshing relatively difficult and promotes domestication. It is said that. Although the sh4 gene has already been isolated and reported, the only knowledge regarding its biological function was related to degranulation.
Li ら(非特許文献１)や Linら（非特許文献２）の報告によると、すべての栽培イネはsh4遺伝子に欠損を持つことで、脱粒性が比較的難になり、栽培化が進んだとされている。sh4遺伝子は既に単離され、報告されているが、その生物学的機能に関する知見は、脱粒性に関するもののみであった。 So far, several genes have been identified for the grain size of plants, but it has not been easy to modify the grain size with or without one known gene.
According to reports of Li et al. (Non-patent Document 1) and Lin et al. (Non-patent Document 2), all cultivated rice has a deficiency in the sh4 gene, which makes threshing relatively difficult and promotes domestication. It is said that. Although the sh4 gene has already been isolated and reported, the only knowledge regarding its biological function was related to degranulation.
なお、本出願の発明に関連する先行技術文献情報を以下に示す。
The prior art document information related to the invention of the present application is shown below.
本発明は、このような状況に鑑みてなされたものであり、その課題は、sh4遺伝子を植物体に導入する工程および、野生イネから発見された機能型sh4遺伝子を発現することで、植物体の穀粒サイズを増大させた植物体を提供することにある。
The present invention has been made in view of such a situation, and the problem is that a plant body can be obtained by introducing a sh4 gene into a plant body and expressing a functional sh4 gene discovered from wild rice. An object of the present invention is to provide a plant having an increased grain size.
本発明者らは、上記の課題を解決するために、植物体の栽培化に関与する遺伝子に関して鋭意研究を行った。本発明者らは、栽培イネに、野生イネ由来の機能型アリルのsh4遺伝子を形質転換により導入することで、新規機能として籾を大きくし、転流も促進し、結果として穀粒サイズが大きくなることを発見した。T0個体についた種子の測定やT1個体についた穂の観察した結果では、籾が大きくなり、玄米重を約1.5倍に増加できることが明らかとなった（図２、図５）。
また、品種日本晴にsh4遺伝子を導入した系統の自殖T1後代4個体(T0-3後代-１、T0-3後代-2、T0-3後代-3、T0-5後代-1)について、人工気象室にてイネ植物体の栽培を行い、穂ごとのモミ重（mｇ）（図７）、個体ごとの総籾数(稔実粒、不稔粒）（図８）、及び個体ごとの穂重（図９）を測定した。その結果、これらの個体は、ベクターコントロール及び日本晴に比べて、有意にモミ重及び個体ごとの穂重（収量）が増加していることが明らかとなった（図７～９）。また、個体ごとの総籾数や穂数には差がなかった（図７、図８）。また、稔性を下げる効果はなく、上げる可能性があるが、この点は、更に解析が必要である（図８）。Ｔ１個体種子・穂の変化は図６に示すように、写真でも顕著である。稔性に関して、今回は、T0-3後代-1やT0-3-後代-2で有意に高く、個体ごとの収量に相当する穂重は、日本晴やベクターコントロールに比して、約2.5倍を示した。
また、他のイネ品種「にこまる」及び「オオチカラ（大力）」に、sh4遺伝子を導入したところ、これらの品種の形質転換当代T0個体において、稔実粒の平均モ ミ重(mg)が有意に増加することが明らかとなった（図１０、図１１）。
即ち、本発明者らは機能型アリルのsh4遺伝子を植物体で発現させることにより、植物体の穀粒サイズを増大させることに成功し、個体辺りの収量が増加することを示し、これにより本発明を完成するに至った。 In order to solve the above-mentioned problems, the present inventors have conducted intensive studies on genes involved in plant cultivation. The present inventors introduced a functional allele sh4 gene derived from wild rice into the cultivated rice by transformation, thereby increasing the culm as a new function and promoting translocation, resulting in a large grain size. I found out that As a result of measuring the seeds attached to the T0 individuals and observing the ears attached to the T1 individuals, it became clear that the wrinkles were increased and the brown rice weight could be increased by about 1.5 times (FIGS. 2 and 5).
In addition, 4 self-breeding T1 progenies (T0-3 progeny-1, T0-3 progeny-2, T0-3 progeny-3, T0-5 progeny-1) of the line that introduced the sh4 gene into the breed Nipponbare were artificially Rice plants are cultivated in the meteorological chamber, fir weight per milligram (mg) (Fig. 7), total number of pods per individual (fruit seeds, sterile grains) (Fig. 8), and ears per individual The weight (Figure 9) was measured. As a result, it was clarified that these individuals had significantly increased fir weight and ear weight (yield) for each individual compared to vector control and Nipponbare (FIGS. 7 to 9). Moreover, there was no difference in the total number of pupae and the number of spikes for each individual (FIGS. 7 and 8). In addition, there is no effect of reducing the inertia, and there is a possibility of increasing this, but this point requires further analysis (FIG. 8). Changes in T1 seeds and ears are also noticeable in photographs as shown in FIG. In terms of fertility, this time it was significantly higher in T0-3 progeny-1 and T0-3-progeny-2, and the ear weight corresponding to the yield of each individual was about 2.5 times that of Nipponbare and vector control. Indicated.
In addition, when sh4 gene was introduced into other rice varieties “Nikomaru” and “Ochikara” (large power), the average fir weight (mg) of cereal grains was significant in the transgenic T0 individuals of these varieties. (Figs. 10 and 11).
That is, the present inventors have succeeded in increasing the grain size of the plant body by expressing the functional allele sh4 gene in the plant body, and that the yield per individual is increased. The invention has been completed.
また、品種日本晴にsh4遺伝子を導入した系統の自殖T1後代4個体(T0-3後代-１、T0-3後代-2、T0-3後代-3、T0-5後代-1)について、人工気象室にてイネ植物体の栽培を行い、穂ごとのモミ重（mｇ）（図７）、個体ごとの総籾数(稔実粒、不稔粒）（図８）、及び個体ごとの穂重（図９）を測定した。その結果、これらの個体は、ベクターコントロール及び日本晴に比べて、有意にモミ重及び個体ごとの穂重（収量）が増加していることが明らかとなった（図７～９）。また、個体ごとの総籾数や穂数には差がなかった（図７、図８）。また、稔性を下げる効果はなく、上げる可能性があるが、この点は、更に解析が必要である（図８）。Ｔ１個体種子・穂の変化は図６に示すように、写真でも顕著である。稔性に関して、今回は、T0-3後代-1やT0-3-後代-2で有意に高く、個体ごとの収量に相当する穂重は、日本晴やベクターコントロールに比して、約2.5倍を示した。
また、他のイネ品種「にこまる」及び「オオチカラ（大力）」に、sh4遺伝子を導入したところ、これらの品種の形質転換当代T0個体において、稔実粒の平均モ ミ重(mg)が有意に増加することが明らかとなった（図１０、図１１）。
即ち、本発明者らは機能型アリルのsh4遺伝子を植物体で発現させることにより、植物体の穀粒サイズを増大させることに成功し、個体辺りの収量が増加することを示し、これにより本発明を完成するに至った。 In order to solve the above-mentioned problems, the present inventors have conducted intensive studies on genes involved in plant cultivation. The present inventors introduced a functional allele sh4 gene derived from wild rice into the cultivated rice by transformation, thereby increasing the culm as a new function and promoting translocation, resulting in a large grain size. I found out that As a result of measuring the seeds attached to the T0 individuals and observing the ears attached to the T1 individuals, it became clear that the wrinkles were increased and the brown rice weight could be increased by about 1.5 times (FIGS. 2 and 5).
In addition, 4 self-breeding T1 progenies (T0-3 progeny-1, T0-3 progeny-2, T0-3 progeny-3, T0-5 progeny-1) of the line that introduced the sh4 gene into the breed Nipponbare were artificially Rice plants are cultivated in the meteorological chamber, fir weight per milligram (mg) (Fig. 7), total number of pods per individual (fruit seeds, sterile grains) (Fig. 8), and ears per individual The weight (Figure 9) was measured. As a result, it was clarified that these individuals had significantly increased fir weight and ear weight (yield) for each individual compared to vector control and Nipponbare (FIGS. 7 to 9). Moreover, there was no difference in the total number of pupae and the number of spikes for each individual (FIGS. 7 and 8). In addition, there is no effect of reducing the inertia, and there is a possibility of increasing this, but this point requires further analysis (FIG. 8). Changes in T1 seeds and ears are also noticeable in photographs as shown in FIG. In terms of fertility, this time it was significantly higher in T0-3 progeny-1 and T0-3-progeny-2, and the ear weight corresponding to the yield of each individual was about 2.5 times that of Nipponbare and vector control. Indicated.
In addition, when sh4 gene was introduced into other rice varieties “Nikomaru” and “Ochikara” (large power), the average fir weight (mg) of cereal grains was significant in the transgenic T0 individuals of these varieties. (Figs. 10 and 11).
That is, the present inventors have succeeded in increasing the grain size of the plant body by expressing the functional allele sh4 gene in the plant body, and that the yield per individual is increased. The invention has been completed.
本発明は、より具体的には以下の（１）～（１３）を提供するものである。
（１）下記（ａ）から（ｄ）のいずれかに記載のDNAを含む、植物体の穀粒の大きさを増大させた植物体。
（ａ）配列番号：３に記載のアミノ酸配列からなるタンパク質をコードするDNA
（ｂ）配列番号：１又は２に記載の塩基配列のコード領域を含むDNA
（ｃ）配列番号：３に記載のアミノ酸配列において１または複数のアミノ酸が置換、欠失、付加、および／または挿入されたアミノ酸配列からなるタンパク質をコードするDNA
（ｄ）配列番号：１又は２に記載の塩基配列からなるDNAとストリンジェントな条件下でハイブリダイズするDNA
（２）前記植物体が単子葉植物である、（１）に記載の植物体。
（３）植物体がイネ科植物である、（１）に記載の植物体。
（４）下記（ａ）から（ｄ）のいずれかに記載のDNAが発現するように導入されたベクター。
（ａ）配列番号：３に記載のアミノ酸配列からなるタンパク質をコードするDNA
（ｂ）配列番号：１又は２に記載の塩基配列のコード領域を含むDNA
（ｃ）配列番号：３に記載のアミノ酸配列において１または複数のアミノ酸が置換、欠失、付加、および／または挿入されたアミノ酸配列からなるタンパク質をコードするDNA
（ｄ）配列番号：１又は２に記載の塩基配列からなるDNAとストリンジェントな条件下でハイブリダイズするDNA
（５）（４）に記載のベクターが導入された宿主細胞。
（６）（４）に記載のベクターが導入された植物細胞。
（７）（６）に記載の植物細胞を含む形質転換植物体。
（８）（７）に記載の形質転換植物体の子孫またはクローンである、形質転換植物体。
（９）（７）または（８）に記載の形質転換植物体の繁殖材料。
（１０）下記（ａ）から（ｄ）のいずれかに記載のDNAを植物体の細胞内で発現させる工程を含む、植物体の穀粒の大きさを増大させる方法。
（ａ）配列番号：３に記載のアミノ酸配列からなるタンパク質をコードするDNA
（ｂ）配列番号：１又は２に記載の塩基配列のコード領域を含むDNA
（ｃ）配列番号：３に記載のアミノ酸配列において１または複数のアミノ酸が置換、欠失、付加、および／または挿入されたアミノ酸配列からなるタンパク質をコードするDNA
（ｄ）配列番号：１又は２に記載の塩基配列からなるDNAとストリンジェントな条件下でハイブリダイズするDNA
（１１）前記植物体が単子葉植物である、（１０）に記載の方法。
（１２）前記植物体がイネ科植物である、（１０）に記載の方法。
（１３）交配により、前記DNAを植物体に導入することを特徴とする、（１０）～（１２）のいずれかに記載の方法。 More specifically, the present invention provides the following (1) to (13).
(1) A plant having an increased grain size of the plant, comprising the DNA according to any one of (a) to (d) below.
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 3
(B) DNA containing the coding region of the base sequence described in SEQ ID NO: 1 or 2
(C) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 3
(D) DNA that hybridizes under stringent conditions with DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1 or 2
(2) The plant according to (1), wherein the plant is a monocotyledonous plant.
(3) The plant body according to (1), wherein the plant body is a gramineous plant.
(4) A vector introduced so that the DNA according to any one of (a) to (d) below is expressed.
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 3
(B) DNA containing the coding region of the base sequence described in SEQ ID NO: 1 or 2
(C) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 3
(D) DNA that hybridizes under stringent conditions with DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1 or 2
(5) A host cell into which the vector according to (4) has been introduced.
(6) A plant cell into which the vector according to (4) is introduced.
(7) A transformed plant comprising the plant cell according to (6).
(8) A transformed plant that is a descendant or clone of the transformed plant according to (7).
(9) A propagation material for the transformed plant according to (7) or (8).
(10) A method for increasing the grain size of a plant comprising the step of expressing the DNA according to any one of (a) to (d) below in a cell of the plant.
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 3
(B) DNA containing the coding region of the base sequence described in SEQ ID NO: 1 or 2
(C) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 3
(D) DNA that hybridizes under stringent conditions with DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1 or 2
(11) The method according to (10), wherein the plant body is a monocotyledonous plant.
(12) The method according to (10), wherein the plant body is a gramineous plant.
(13) The method according to any one of (10) to (12), wherein the DNA is introduced into a plant body by crossing.
（１）下記（ａ）から（ｄ）のいずれかに記載のDNAを含む、植物体の穀粒の大きさを増大させた植物体。
（ａ）配列番号：３に記載のアミノ酸配列からなるタンパク質をコードするDNA
（ｂ）配列番号：１又は２に記載の塩基配列のコード領域を含むDNA
（ｃ）配列番号：３に記載のアミノ酸配列において１または複数のアミノ酸が置換、欠失、付加、および／または挿入されたアミノ酸配列からなるタンパク質をコードするDNA
（ｄ）配列番号：１又は２に記載の塩基配列からなるDNAとストリンジェントな条件下でハイブリダイズするDNA
（２）前記植物体が単子葉植物である、（１）に記載の植物体。
（３）植物体がイネ科植物である、（１）に記載の植物体。
（４）下記（ａ）から（ｄ）のいずれかに記載のDNAが発現するように導入されたベクター。
（ａ）配列番号：３に記載のアミノ酸配列からなるタンパク質をコードするDNA
（ｂ）配列番号：１又は２に記載の塩基配列のコード領域を含むDNA
（ｃ）配列番号：３に記載のアミノ酸配列において１または複数のアミノ酸が置換、欠失、付加、および／または挿入されたアミノ酸配列からなるタンパク質をコードするDNA
（ｄ）配列番号：１又は２に記載の塩基配列からなるDNAとストリンジェントな条件下でハイブリダイズするDNA
（５）（４）に記載のベクターが導入された宿主細胞。
（６）（４）に記載のベクターが導入された植物細胞。
（７）（６）に記載の植物細胞を含む形質転換植物体。
（８）（７）に記載の形質転換植物体の子孫またはクローンである、形質転換植物体。
（９）（７）または（８）に記載の形質転換植物体の繁殖材料。
（１０）下記（ａ）から（ｄ）のいずれかに記載のDNAを植物体の細胞内で発現させる工程を含む、植物体の穀粒の大きさを増大させる方法。
（ａ）配列番号：３に記載のアミノ酸配列からなるタンパク質をコードするDNA
（ｂ）配列番号：１又は２に記載の塩基配列のコード領域を含むDNA
（ｃ）配列番号：３に記載のアミノ酸配列において１または複数のアミノ酸が置換、欠失、付加、および／または挿入されたアミノ酸配列からなるタンパク質をコードするDNA
（ｄ）配列番号：１又は２に記載の塩基配列からなるDNAとストリンジェントな条件下でハイブリダイズするDNA
（１１）前記植物体が単子葉植物である、（１０）に記載の方法。
（１２）前記植物体がイネ科植物である、（１０）に記載の方法。
（１３）交配により、前記DNAを植物体に導入することを特徴とする、（１０）～（１２）のいずれかに記載の方法。 More specifically, the present invention provides the following (1) to (13).
(1) A plant having an increased grain size of the plant, comprising the DNA according to any one of (a) to (d) below.
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 3
(B) DNA containing the coding region of the base sequence described in SEQ ID NO: 1 or 2
(C) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 3
(D) DNA that hybridizes under stringent conditions with DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1 or 2
(2) The plant according to (1), wherein the plant is a monocotyledonous plant.
(3) The plant body according to (1), wherein the plant body is a gramineous plant.
(4) A vector introduced so that the DNA according to any one of (a) to (d) below is expressed.
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 3
(B) DNA containing the coding region of the base sequence described in SEQ ID NO: 1 or 2
(C) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 3
(D) DNA that hybridizes under stringent conditions with DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1 or 2
(5) A host cell into which the vector according to (4) has been introduced.
(6) A plant cell into which the vector according to (4) is introduced.
(7) A transformed plant comprising the plant cell according to (6).
(8) A transformed plant that is a descendant or clone of the transformed plant according to (7).
(9) A propagation material for the transformed plant according to (7) or (8).
(10) A method for increasing the grain size of a plant comprising the step of expressing the DNA according to any one of (a) to (d) below in a cell of the plant.
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 3
(B) DNA containing the coding region of the base sequence described in SEQ ID NO: 1 or 2
(C) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 3
(D) DNA that hybridizes under stringent conditions with DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1 or 2
(11) The method according to (10), wherein the plant body is a monocotyledonous plant.
(12) The method according to (10), wherein the plant body is a gramineous plant.
(13) The method according to any one of (10) to (12), wherein the DNA is introduced into a plant body by crossing.
本発明は、機能型sh4遺伝子を植物体に導入する、又は野生イネから発見された機能型sh4遺伝子を発現することで、植物体の穀粒サイズを増大させた植物体を提供することにある。
An object of the present invention is to provide a plant in which the grain size of the plant body is increased by introducing the functional sh4 gene into the plant body or expressing the functional sh4 gene discovered from wild rice. .
本発明のsh4遺伝子は、複数の論文発表によると、すべての栽培イネが機能欠損を起こしている遺伝子である。イネの栽培化の初期過程で、栽培しやすくするために、脱粒性を軽減することを目的に、機能欠損型を古代の人類が選抜したことで栽培イネが確立されたと考えられ、栽培イネに機能型アリルを持つものは存在しないと考えられる。今回、機能型アリルのsh4遺伝子が、植物体の穀粒の大きさを増大させる作用を有していることから、該タンパク質をコードするDNAで植物を形質転換することにより、穀粒の大きさが増大した植物体の育成が可能である。また、単純な考察から、交配により、野生イネから機能型アリルのsh4遺伝子を導入した準同質置換系統でも、同様な効果が期待できる。
The sh4 gene of the present invention is a gene in which all cultivated rice is deficient in function, according to multiple paper publications. It is considered that cultivated rice was established by the selection of a function-deficient type by ancient humans with the aim of reducing threshing properties in order to make it easier to cultivate in the initial process of rice cultivation. It is considered that there is no functional allele. This time, the functional allele sh4 gene has the effect of increasing the grain size of the plant body, so by transforming the plant with DNA encoding the protein, It is possible to grow plants with increased From simple considerations, a similar effect can be expected even in a near-isogenic replacement line in which a functional allele sh4 gene is introduced from wild rice by crossing.
本発明において、sh4遺伝子が導入される植物は特に限定されるものではないが、好ましくは単子葉植物であり、より好ましくはイネ科植物であり、最も好ましくは栽培イネである。本発明において、イネ科植物の品種は特に限定されるものではないが、好ましくは「日本晴」「にこまる」又は「オオチカラ（大力）」等を挙げることが出来る。
In the present invention, the plant into which the sh4 gene is introduced is not particularly limited, but is preferably a monocotyledonous plant, more preferably a gramineous plant, and most preferably cultivated rice. In the present invention, the varieties of gramineous plants are not particularly limited, but preferred examples include “Nipponbare”, “Nikomaru”, “Ochikara (great power)” and the like.
本発明において、「植物体の穀粒の大きさを増大させる」とは、本発明のsh4遺伝子を植物内で発現させることにより、収穫時の穀粒の体積や重量を増大させることをいう。また、籾の大きさを増大させる効果や転流を促進する効果も「植物体の穀粒の大きさを増大させる」ことに相当する。
穀粒の大きさの増大効果は、植物の穀粒の発生過程のみに発現する効果であってもよい。
また、全ての穀粒において増大効果がみられるものであってもよいし、ある特定の穀粒にのみ増大効果がみられるものであってもよい。
本発明において、「植物体の穀粒の大きさが増大したかいなか」については、穂ごとのモミ重（mｇ）又は個体ごとの穂重（g）を測定することにより確認することができる。 In the present invention, “to increase the grain size of a plant” means to increase the volume and weight of the grain at the time of harvest by expressing the sh4 gene of the present invention in the plant. Moreover, the effect of increasing the size of the cocoon and the effect of promoting commutation also correspond to “increasing the size of the kernel of the plant”.
The effect of increasing the size of the grain may be an effect that appears only in the process of generating the kernel of the plant.
Moreover, the increase effect may be seen in all the grains, or the increase effect may be seen only in a specific grain.
In the present invention, “whether the grain size of the plant body has increased” can be confirmed by measuring the fir weight (mg) per ear or the ear weight (g) per individual.
穀粒の大きさの増大効果は、植物の穀粒の発生過程のみに発現する効果であってもよい。
また、全ての穀粒において増大効果がみられるものであってもよいし、ある特定の穀粒にのみ増大効果がみられるものであってもよい。
本発明において、「植物体の穀粒の大きさが増大したかいなか」については、穂ごとのモミ重（mｇ）又は個体ごとの穂重（g）を測定することにより確認することができる。 In the present invention, “to increase the grain size of a plant” means to increase the volume and weight of the grain at the time of harvest by expressing the sh4 gene of the present invention in the plant. Moreover, the effect of increasing the size of the cocoon and the effect of promoting commutation also correspond to “increasing the size of the kernel of the plant”.
The effect of increasing the size of the grain may be an effect that appears only in the process of generating the kernel of the plant.
Moreover, the increase effect may be seen in all the grains, or the increase effect may be seen only in a specific grain.
In the present invention, “whether the grain size of the plant body has increased” can be confirmed by measuring the fir weight (mg) per ear or the ear weight (g) per individual.
本発明に用いられる機能型sh4遺伝子のゲノムDNAの塩基配列を配列番号：１に、該遺伝子のORF領域の塩基配列を配列番号：２に、及び該DNAがコードするタンパク質のアミノ酸配列を配列番号：３に示す。又、機能欠失型sh4タンパク質のアミノ酸配列を配列番号：４に示す。
The nucleotide sequence of the genomic DNA of the functional sh4 gene used in the present invention is SEQ ID NO: 1, the nucleotide sequence of the ORF region of the gene is SEQ ID NO: 2, and the amino acid sequence of the protein encoded by the DNA is SEQ ID NO: : Shown in 3. The amino acid sequence of the function-deficient sh4 protein is shown in SEQ ID NO: 4.
本発明に用いられるDNAには、交配により移される染色体断片中のゲノムDNA、ゲノムDNA、cDNA、および化学合成DNAが含まれる。ゲノムDNAおよびcDNAの調製は、当業者にとって常套手段を利用して行うことが可能である。ゲノムDNAは、例えば、本発明のsh4遺伝子を有するイネ品種からゲノムDNAを抽出し、ゲノミックライブラリー（ベクターとしては、プラスミド、ファージ、コスミド、BAC、PACなどが利用できる）を作成し、これを展開して、本発明のsh4タンパク質をコードするDNA（例えば、配列番号：２）を基に調製したプローブを用いてコロニーハイブリダイゼーションあるいはプラークハイブリダイゼーションを行うことにより調製することが可能である。また、本発明のsh4タンパク質をコードするDNA（例えば、配列番号：２）に特異的なプライマーを作成し、これを利用したPCRをおこなうことによって調製することも可能である。また、cDNAは、例えば、本発明のsh4遺伝子を有するイネ品種から抽出したmRNAを基にcDNAを合成し、これをλZAP等のベクターに挿入してcDNAライブラリーを作成し、これを展開して、上記と同様にコロニーハイブリダイゼーションあるいはプラークハイブリダイゼーションを行うことにより、また、PCRを行うことにより調製することが可能である。
The DNA used in the present invention includes genomic DNA, genomic DNA, cDNA, and chemically synthesized DNA in chromosome fragments transferred by mating. 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 rice varieties having the sh4 gene of the present invention, and a genomic library (plasmid, phage, cosmid, BAC, PAC, etc. can be used as a vector) It can be prepared by developing and performing colony hybridization or plaque hybridization using a probe prepared based on the DNA encoding the sh4 protein of the present invention (for example, SEQ ID NO: 2). It is also possible to prepare a primer specific for the DNA encoding the sh4 protein of the present invention (for example, SEQ ID NO: 2) and perform PCR using this primer. In addition, for example, cDNA is synthesized based on mRNA extracted from rice varieties having the sh4 gene of the present invention, and inserted into a vector such as λZAP to create a cDNA library. It can be prepared by performing colony hybridization or plaque hybridization in the same manner as described above, or by performing PCR.
本発明に用いられるDNAは、配列番号：３に記載の機能型sh4タンパク質と機能的に同等なタンパク質をコードするDNAを包含する。ここで「sh4タンパク質と同等の機能を有する」とは、対象となるタンパク質が植物体の穀粒の大きさを増大させる機能を有することを指す。このようなDNAは、好ましくは単子葉植物由来であり、より好ましくはイネ科植物由来であり、最も好ましくは現在の野生イネ由来である。
The DNA used in the present invention includes DNA encoding a protein functionally equivalent to the functional sh4 protein described in SEQ ID NO: 3. Here, “having a function equivalent to the sh4 protein” means that the target protein has a function of increasing the grain size of the plant body. Such DNA is preferably derived from monocotyledonous plants, more preferably from gramineous plants, and most preferably from current wild rice.
このようなDNAには、例えば、配列番号：３に記載のアミノ酸配列において１若しくは複数のアミノ酸が置換、欠失、付加および/または挿入されたアミノ酸配列からなるタンパク質をコードする変異体、誘導体、アレル、バリアントおよびホモログが含まれる。
Such DNA includes, for example, mutants, derivatives, and the like that encode proteins consisting of amino acid sequences in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence set forth in SEQ ID NO: 3. Alleles, variants and homologs are included.
アミノ酸配列が改変されたタンパク質をコードするDNAを調製するための当業者によく知られた方法としては、例えば、site-directed mutagenesis法が挙げられる。また、塩基配列の変異によりコードするタンパク質のアミノ酸配列が変異することは、自然界においても生じ得る。このように天然機能型のsh4タンパク質をコードするアミノ酸配列において1もしくは複数のアミノ酸が置換、欠失もしくは付加したアミノ酸配列を有するタンパク質をコードするDNAであっても、天然機能型のsh4タンパク質（配列番号：３）と同等の機能を有するタンパク質をコードする限り、本発明のDNAに含まれる。また、たとえ、塩基配列が変異した場合でも、それがタンパク質中のアミノ酸の変異を伴わない場合（縮重変異）もあり、このような縮重変異体も本発明のDNAに含まれる。
As a method well known to those skilled in the art for preparing a DNA encoding a protein having a modified amino acid sequence, for example, a site-directed mutagenesis method can be mentioned. In addition, the amino acid sequence of the encoded protein may be mutated in nature due to the mutation of the base sequence. Thus, even if a DNA encoding a protein having an amino acid sequence in which one or a plurality of amino acids are substituted, deleted or added in the amino acid sequence encoding the natural functional sh4 protein, the natural functional sh4 protein (sequence) As long as it encodes a protein having a function equivalent to that of No. 3), it is included in the DNA of the present invention. Moreover, even if the base sequence is mutated, it may not be accompanied by amino acid mutation in the protein (degenerate mutation), and such a degenerate mutant is also included in the DNA of the present invention.
あるDNAが植物体の穀粒の大きさを増大させる機能を有するタンパク質をコードするか否かは以下のようにして評価することができる。最も一般的な方法としては、該DNAが導入された植物体の穀粒の大きさを調べる手法である。植物体の穀粒の大きさが増大した場合には、導入したDNAが植物体の穀粒の大きさを増大させる機能を有するタンパク質をコードしていることが分かる。
Whether or not a certain DNA encodes a protein having a function of increasing the grain size of a plant body can be evaluated as follows. The most general method is a method for examining the grain size of the plant body into which the DNA has been introduced. When the grain size of the plant body is increased, it can be seen that the introduced DNA encodes a protein having a function of increasing the grain size of the plant body.
配列番号：３に記載のsh4タンパク質と機能的に同等なタンパク質をコードするDNAを調製するために、当業者によく知られた他の方法としては、ハイブリダイゼーション技術やポリメラーゼ連鎖反応（PCR）技術を利用する方法が挙げられる。即ち、当業者にとっては、sh4遺伝子の塩基配列（配列番号：１又は２）もしくはその一部をプローブとして、またsh4遺伝子（配列番号：１又は２）に特異的にハイブリダイズするオリゴヌクレオチドをプライマーとして、イネや他の植物からsh4遺伝子と高い相同性を有するDNAを単離することは通常行いうることである。このようにハイブリダイズ技術やPCR技術により単離しうるsh4タンパク質と同等の機能を有するタンパク質をコードするDNAもまた本発明のDNAに含まれる。
Other methods well known to those skilled in the art for preparing DNA encoding a protein functionally equivalent to the sh4 protein described in SEQ ID NO: 3 include hybridization techniques and polymerase chain reaction (PCR) techniques. The method of using is mentioned. That is, for those skilled in the art, the nucleotide sequence of the sh4 gene (SEQ ID NO: 1 or 2) or a part thereof is used as a probe, and an oligonucleotide that specifically hybridizes to the sh4 gene (SEQ ID NO: 1 or 2) is used as a primer. Thus, it is usually possible to isolate DNA having high homology with the sh4 gene from rice and other plants. Thus, DNA encoding a protein having a function equivalent to that of sh4 protein that can be isolated by hybridization technology or PCR technology is also included in the DNA of the present invention.
このようなDNAを単離するためには、好ましくはストリンジェントな条件下でハイブリダイゼーション反応を行う。本発明においてストリンジェントなハイブリダイゼーション条件とは、6M尿素、 0.4%SDS、0.5×SSCの条件またはこれと同等のストリンジェンシーのハイブリダイゼーション条件を指す。よりストリンジェンシーの高い条件、例えば、6M尿素、0.4%SDS、0.1×SSCの条件を用いることにより、より相同性の高いDNAの単離を期待することができる。これにより単離されたDNAは、アミノ酸レベルにおいて、sh4タンパク質のアミノ酸配列（配列番号：３）と高い相同性を有すると考えられる。高い相同性とは、アミノ酸配列全体で、少なくとも50％以上、さらに好ましくは70％以上、さらに好ましくは90％以上（例えば、95%,96%,97%,98%,99%以上）の配列の同一性を指す。アミノ酸配列や塩基配列の同一性は、カーリンおよびアルチュールによるアルゴリズムBLAST（Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990、Proc Natl Acad Sci USA 90: 5873, 1993）を用いて決定できる。BLASTのアルゴリズムに基づいたBLASTNやBLASTXと呼ばれるプログラムが開発されている（Altschul SF, et al: J Mol Biol 215: 403, 1990）。BLASTNを用いて塩基配列を解析する場合は、パラメーターは、例えばscore＝100、wordlength＝12とする。また、BLASTXを用いてアミノ酸配列を解析する場合は、パラメーターは、例えばscore＝50、wordlength＝3とする。BLASTとGapped BLASTプログラムを用いる場合は、各プログラムのデフォルトパラメーターを用いる。これらの解析方法の具体的な手法は公知である。
In order to isolate such DNA, a hybridization reaction is preferably performed under stringent conditions. In the present invention, stringent hybridization conditions refer to conditions of 6M urea, 0.4% SDS, 0.5 × SSC, or equivalent stringency hybridization conditions. Isolation of DNA with higher homology can be expected by using conditions with higher stringency, for example, conditions of 6M urea, 0.4% SDS, 0.1 × SSC. The isolated DNA is considered to have high homology with the amino acid sequence of the sh4 protein (SEQ ID NO: 3) at the amino acid level. High homology means a sequence of at least 50% or more, more preferably 70% or more, more preferably 90% or more (for example, 95%, 96%, 97%, 98%, 99% or more) in the entire amino acid sequence. Refers to the identity of The identity of amino acid sequences and base sequences is determined using the algorithm BLAST (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990, Proc Natl Acad Sci USA 90: 5873, 1993) by Carlin and Arthur it can. Programs called BLASTN and BLASTX based on the BLAST algorithm have been developed (Altschul SF, et al: J Mol Biol 215: 403, 1990). When analyzing a base sequence using BLASTN, parameters are set to, for example, score = 100 and wordlength = 12. In addition, when an amino acid sequence is analyzed using BLASTX, the parameters are, for example, score = 50 and wordlength = 3. When using BLAST and Gapped BLAST programs, use the default parameters of each program. Specific methods of these analysis methods are known.
本発明は、野生イネと栽培イネの交配後代から、sh4遺伝子座が野生イネの機能型アリルをもつ個体を選抜し、穀粒サイズが母本より大きい植物体、その後代、固定系統、品種等を提供できる。野生アリルの遺伝子の導入は、穀粒サイズの増加と、野生イネsh4アリルを持つことを両方満たすということで、特許性を確認することができる。野生イネアリルを持っているかどうかは、報告のある機能欠損アミノ酸部位が機能型アミノ酸に変化していることを、特定ゲノム部位をPCR等で増幅し、DNA配列のシークエンス既存の技術で確認する等で、確認可能である。
The present invention selects individuals having a functional allele of wild rice from the mating progeny of wild rice and cultivated rice, and a plant body whose grain size is larger than the parent, progeny, fixed line, variety, etc. Can provide. The introduction of the wild allele gene can confirm the patentability by satisfying both the increase in grain size and having the wild rice sh4 allele. Whether or not it has wild rice alleles can be confirmed by amplifying a specific genomic site by PCR, etc., and confirming the sequence of the DNA sequence using existing technology Can be confirmed.
また、本発明のDNAを利用して植物体の穀粒の大きさを増大した形質転換植物体も提供できる。この形質転換植物体を作製する場合には、本発明のタンパク質をコードするDNA（制御領域を含んでもいい）を適当なベクターに挿入して、これを植物細胞に導入し、これにより得られた形質転換植物細胞を再生させる。本発明者等により単離されたsh4遺伝子は、植物体の穀粒の大きさを増大させる作用を有するが、このsh4遺伝子を任意の品種に導入し過剰に発現させることによりそれらの系統の穀粒の大きさを増大させることが可能である。この形質転換に要する期間は、従来のような交配による遺伝子移入に比較して極めて短期間であり、また、他の形質の変化を伴わない点で有利である。
In addition, a transformed plant body in which the grain size of the plant body is increased using the DNA of the present invention can also be provided. When producing this transformed plant body, DNA (which may contain a control region) encoding the protein of the present invention was inserted into an appropriate vector, which was introduced into plant cells, and thus obtained. Regenerate transformed plant cells. The sh4 gene isolated by the present inventors has an effect of increasing the grain size of the plant body, but by introducing this sh4 gene into an arbitrary variety and overexpressing it, the grains of those lines It is possible to increase the size of the grains. The period required for this transformation is extremely short as compared with conventional gene transfer by crossing, and is advantageous in that it does not involve any other changes in traits.
また、本発明は、上記本発明のDNAが挿入されたベクターを提供する。本発明のベクターとしては、形質転換植物体作製のために植物細胞内で本発明のDNAを発現させるためのベクターを挙げることができる。このようなベクターとしては、植物細胞で転写可能なプロモーター配列と転写産物の安定化に必要なポリアデニレーション部位を含むターミネーター配列を含んでいれば特に制限されず、例えば、プラスミド「pBI121」、「pBI221」、「pBI101」（いずれもClontech社製）などが挙げられる。植物細胞の形質転換に用いられるベクターとしては、該細胞内で挿入遺伝子を発現させることが可能なものであれば特に制限はない。例えば、植物細胞内での恒常的な遺伝子発現を行うためのプロモーター（例えば、カリフラワーモザイクウイルスの35Sプロモーター）を有するベクターや外的な刺激により誘導的に活性化されるプロモーターを有するベクターを用いることも可能である。ここでいう「植物細胞」には、種々の形態の植物細胞、例えば、懸濁培養細胞、プロトプラスト、葉の切片、カルスなどが含まれる。
The present invention also provides a vector into which the DNA of the present invention is inserted. Examples of the vector of the present invention include a vector for expressing the DNA of the present invention in a plant cell for producing a transformed plant body. Such a vector is not particularly limited as long as it contains a promoter sequence that can be transcribed in plant cells and a terminator sequence including a polyadenylation site necessary for the stabilization of the transcript. For example, plasmids `` pBI121 '', `` pBI221 "," pBI101 "(both manufactured by Clontech). The vector used for the transformation of plant cells is not particularly limited as long as the inserted gene can be expressed in the cells. For example, using a vector having a promoter (eg, cauliflower mosaic virus 35S promoter) for constitutive gene expression in plant cells or a vector having a promoter inducibly activated by an external stimulus Is also possible. The “plant cell” as used herein includes various forms of plant cells, such as suspension culture cells, protoplasts, leaf sections, and callus.
本発明のベクターは、sh4遺伝子本来のプロモーターはもちろん、本発明のタンパク質を恒常的または誘導的に発現させるためのプロモーターを含有しうる。恒常的に発現させるためのプロモーターとしては、例えば、カリフラワーモザイクウイルスの35Sプロモーター、イネのアクチンプロモーター、トウモロコシのユビキチンプロモーターなどが挙げられる。
The vector of the present invention may contain a promoter for constitutively or inducibly expressing the protein of the present invention as well as the promoter inherent to the sh4 gene. Examples of promoters for constant expression include cauliflower mosaic virus 35S promoter, rice actin promoter, maize ubiquitin promoter, and the like.
また、誘導的に発現させるためのプロモーターとしては、例えば糸状菌・細菌・ウイルスの感染や侵入、低温、高温、乾燥、紫外線の照射、特定の化合物の散布などの外因によって発現することが知られているプロモーターなどが挙げられる。このようなプロモーターとしては、例えば、糸状菌・細菌・ウイルスの感染や侵入によって発現するイネキチナーゼ遺伝子のプロモーターやタバコのPRタンパク質遺伝子のプロモーター、低温によって誘導されるイネの「lip19」遺伝子のプロモーター、高温によって誘導されるイネの「hsp80」遺伝子と「hsp72」遺伝子のプロモーター、乾燥によって誘導されるシロイヌナズナの「rab16」遺伝子のプロモーター、紫外線の照射によって誘導されるパセリのカルコン合成酵素遺伝子のプロモーター、嫌気的条件で誘導されるトウモロコシのアルコールデヒドロゲナーゼ遺伝子のプロモーターなどが挙げられる。また、イネキチナーゼ遺伝子のプロモーターとタバコのPRタンパク質遺伝子のプロモーターはサリチル酸などの特定の化合物によって、「rab16」は植物ホルモンのアブシジン酸の散布によっても誘導される。
In addition, promoters for inducible expression are known to be expressed by external factors such as infection and invasion of filamentous fungi, bacteria, and viruses, low temperature, high temperature, drying, ultraviolet irradiation, and spraying of specific compounds. Promoters and the like. Examples of such promoters include, for example, rice chitinase gene promoters expressed by infection and invasion of filamentous fungi, bacteria and viruses, tobacco PR protein gene promoters, rice lip19 gene promoters induced by low temperature, Rice "hsp80" and "hsp72" gene promoters induced by high temperature, Arabidopsis thaliana "rab16" gene promoter induced by drying, Parsley chalcone synthase gene promoter induced by UV irradiation, Anaerobic And the promoter of corn alcohol dehydrogenase gene induced under a certain condition. The rice chitinase gene promoter and tobacco PR protein gene promoter are also induced by specific compounds such as salicylic acid, and “rab16” is also induced by spraying the plant hormone abscisic acid.
また、本発明は、本発明のベクターが導入された形質転換細胞を提供する。本発明のベクターが導入される細胞には、形質転換植物体作製のための植物細胞が含まれる。植物細胞としては特に制限はなく、例えば、イネ、シロイヌナズナ、トウモロコシ、ジャガイモ、タバコなどの細胞が挙げられる。本発明の植物細胞には、培養細胞の他、植物体中の細胞も含まれる。また、プロトプラスト、苗条原基、多芽体、毛状根も含まれる。植物細胞へのベクターの導入は、ポリエチレングリコール法、電気穿孔法（エレクトロポーレーション）、アグロバクテリウムを介する方法、パーティクルガン法など当業者に公知の種々の方法を用いることができる。形質転換植物細胞からの植物体の再生は、植物細胞の種類に応じて当業者に公知の方法で行うことが可能である。例えば、イネにおいては、形質転換植物体を作出する手法については、ポリエチレングリコールによりプロトプラストへ遺伝子導入し、植物体（インド型イネ品種が適している）を再生させる方法、電気パルスによりプロトプラストへ遺伝子導入し、植物体（日本型イネ品種が適している）を再生させる方法、パーティクルガン法により細胞へ遺伝子を直接導入し、植物体を再生させる方法、およびアグロバクテリウムを介して遺伝子を導入し、植物体を再生させる方法など、いくつかの技術が既に確立し、本願発明の技術分野において広く用いられている。本発明においては、これらの方法を好適に用いることができる。
The present invention also provides a transformed cell into which the vector of the present invention has been introduced. The cells into which the vector of the present invention is introduced include plant cells for producing transformed plants. There is no restriction | limiting in particular as a plant cell, For example, cells, such as a rice, Arabidopsis thaliana, corn, a potato, a tobacco, are mentioned. The plant cells of the present invention include cultured cells as well as cells in the plant body. Also included are protoplasts, shoot primordia, multi-buds, and hairy roots. For introduction of a vector into a plant cell, various methods known to those skilled in the art such as polyethylene glycol method, electroporation (electroporation), Agrobacterium-mediated method, and particle gun method can be used. Regeneration of plant bodies from transformed plant cells can be performed by methods known to those skilled in the art depending on the type of plant cells. For example, in rice, methods for producing transformed plants include gene transfer into protoplasts using polyethylene glycol and regeneration of plants (suitable for Indian rice varieties), gene transfer into protoplasts using electric pulses The method of regenerating plants (Japanese rice varieties are suitable), the method of directly introducing genes into cells by the particle gun method, the method of regenerating plants, and the introduction of genes via Agrobacterium, Several techniques, such as a method for regenerating plant bodies, 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.
形質転換された植物細胞は、再分化させることにより植物体を再生させることが可能である。再分化の方法は植物細胞の種類により異なるが、例えば、イネであればFujimuraら（Plant Tissue Culture Lett. 2:74 (1995)）の方法が挙げられ、トウモロコシであればShillitoら（Bio/Technology 7:581 (1989)）の方法やGorden-Kammら（Plant Cell 2:603(1990)）が挙げられ、ジャガイモであればVisserら（Theor.Appl.Genet 78:594 (1989)）の方法が挙げられ、タバコであればNagataとTakebe（Planta 99:12(1971)）の方法が挙げられ、シロイヌナズナであればAkamaら（Plant Cell Reports12:7-11 (1992)）の方法が挙げられ、ユーカリであれば土肥ら（特開平8-89113号公報）の方法が挙げられる。
The transformed plant cell can regenerate the plant body by redifferentiation. The method of redifferentiation varies depending on the type of plant cell. For example, for rice, the method of Fujimura et al. (Plant Tissue Culture Lett. 2:74 (1995)) can be mentioned, and for maize, Shillito et al. (Bio / Technology 7: 581 (1989)) and Gorden-Kamm et al. (Plant Cell 2: 603 (1990)). For potatoes, Visser et al. (Theor. Appl. Genet 78: 594 (1989)) In the case of tobacco, the method of Nagata and Takebe (Planta 99:12 (1971)) is mentioned, and in the case of Arabidopsis, the method of Akama et al. (Plant Cell Reports12: 7-11 (1992)) is mentioned, and eucalyptus Then, the method of Toi et al. (Japanese Patent Laid-Open No. 8-89113) can be mentioned.
一旦、ゲノム内に本発明のDNAが導入された形質転換植物体が得られれば、該植物体から有性生殖または無性生殖により子孫を得ることが可能である。また、該植物体やその子孫あるいはクローンから繁殖材料（例えば、種子、果実、切穂、塊茎、塊根、株、カルス、プロトプラスト等）を得て、それらを基に該植物体を量産することも可能である。本発明には、本発明のDNAが導入された植物細胞、該細胞を含む植物体、該植物体の子孫およびクローン、並びに該植物体、その子孫、およびクローンの繁殖材料が含まれる。
Once a transformed plant into which the DNA of the present invention has been introduced into the genome is obtained, offspring can be obtained from the plant by sexual reproduction or asexual reproduction. It is also possible to obtain a propagation material (for example, seeds, fruits, cuttings, tubers, tuberous roots, strains, callus, protoplasts, etc.) from the plant body, its descendants or clones, and mass-produce the plant body based on them. Is possible. The present invention includes a plant cell into which the DNA of the present invention has been introduced, a plant containing the cell, progeny and clones of the plant, and propagation material of the plant, its progeny and clones.
このようにして作出された穀粒の大きさが増大した植物体は、野生型植物体と比較して、穀物の収穫量が上がるものと考えられる。本発明の手法を用いれば、農作物の生産性向上に繋がるものと考えられる。
なお本明細書において引用された全ての先行技術文献は、参照として本明細書に組み入れられる。 It is considered that a plant body having a large grain size produced in this way has a higher crop yield than a wild-type plant body. If the method of this invention is used, it will be thought that it leads to the productivity improvement of agricultural products.
It should be noted that all prior art documents cited in the present specification are incorporated herein by reference.
なお本明細書において引用された全ての先行技術文献は、参照として本明細書に組み入れられる。 It is considered that a plant body having a large grain size produced in this way has a higher crop yield than a wild-type plant body. If the method of this invention is used, it will be thought that it leads to the productivity improvement of agricultural products.
It should be noted that all prior art documents cited in the present specification are incorporated herein by reference.
以下、本発明を実施例によりさらに具体的に説明するが本発明はこれら実施例に制限されるものではない。
〔実施例１〕
Ａゲノムを持つ野生イネOryza nivaraからゲノムDNAを抽出し、BACライブラリーを作成した。そのライブラリーから、sh4遺伝子領域のゲノム断片を持つBACクローンをPCRでsh4領域だけを増やす特異的なプライマーを設計し、PCRでの増幅の有無で、sh4遺伝子領域を持つBACクローンを単離、その後、pUC18ベクターにBACクローンDNAを数kbpに短く断片化したDNAをサブクローニングした上で、個々のサブクローンの端読みDNA配列を決定し、アセンブルすることで、O.nivaraのsh4遺伝子ゲノム領域のDNA配列を決定した。得られたsh4領域のゲノム断片配列情報から、既知のsh4遺伝子産物の情報から、プロモーター領域等を予想し、KpnIとBamHI制限酵素による消化反応で、sh4遺伝子領域、約8.8kbp長を切り出し、pPZP２H-lacベクターにそのゲノム断片を導入し、形質転換用のコンストラクトを作成した。そして、単離した機能型アリルのsh4遺伝子のコード領域および制御領域を含む約8.8kbのゲノム断片（図１、配列番号：１）を2つのイネ系統、日本晴、NIL(qSH1) (Konishi et al. 2006)に、イネの形質転換法で、単子葉植物の超迅速形質転換法（日本国特許3141084号）を用いて、遺伝子導入した。形質転換の選抜には、抗生物質であるハイグロマイシンを利用した。それぞれ独立な形質転換系統を１０系統前後作成し、籾重、脱粒性等、各種形質を測定した。 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]
Genomic DNA was extracted from wild rice Oryza nivara with A genome, and BAC library was created. From the library, BAC clones with sh4 gene region genomic fragments were designed with specific primers to increase only the sh4 region by PCR, and BAC clones with sh4 gene region were isolated with or without amplification by PCR, Then, after subcloning the DNA obtained by fragmenting the BAC clone DNA to a few kbp into the pUC18 vector, the end reading DNA sequence of each subclone was determined and assembled, so that the sh4 gene genomic region of O.nivara The DNA sequence was determined. From the obtained genomic fragment sequence information of the sh4 region, the promoter region is predicted from the information of the known sh4 gene product, and the sh4 gene region, about 8.8 kbp length is excised by digestion reaction with KpnI and BamHI restriction enzyme, pPZP2H The genomic fragment was introduced into the -lac vector to create a transformation construct. Then, an approximately 8.8 kb genomic fragment (FIG. 1, SEQ ID NO: 1) containing the coding region and the regulatory region of the isolated functional allele sh4 gene was transformed into two rice lines, Nipponbare, NIL (qSH1) (Konishi et al In 2006, the gene was introduced using the ultra-rapid transformation method of monocotyledons (Japanese Patent No. 314084) by the rice transformation method. Antibiotic hygromycin was used for selection of transformation. About 10 independent transformants were prepared, and various traits such as weight and shedding were measured.
〔実施例１〕
Ａゲノムを持つ野生イネOryza nivaraからゲノムDNAを抽出し、BACライブラリーを作成した。そのライブラリーから、sh4遺伝子領域のゲノム断片を持つBACクローンをPCRでsh4領域だけを増やす特異的なプライマーを設計し、PCRでの増幅の有無で、sh4遺伝子領域を持つBACクローンを単離、その後、pUC18ベクターにBACクローンDNAを数kbpに短く断片化したDNAをサブクローニングした上で、個々のサブクローンの端読みDNA配列を決定し、アセンブルすることで、O.nivaraのsh4遺伝子ゲノム領域のDNA配列を決定した。得られたsh4領域のゲノム断片配列情報から、既知のsh4遺伝子産物の情報から、プロモーター領域等を予想し、KpnIとBamHI制限酵素による消化反応で、sh4遺伝子領域、約8.8kbp長を切り出し、pPZP２H-lacベクターにそのゲノム断片を導入し、形質転換用のコンストラクトを作成した。そして、単離した機能型アリルのsh4遺伝子のコード領域および制御領域を含む約8.8kbのゲノム断片（図１、配列番号：１）を2つのイネ系統、日本晴、NIL(qSH1) (Konishi et al. 2006)に、イネの形質転換法で、単子葉植物の超迅速形質転換法（日本国特許3141084号）を用いて、遺伝子導入した。形質転換の選抜には、抗生物質であるハイグロマイシンを利用した。それぞれ独立な形質転換系統を１０系統前後作成し、籾重、脱粒性等、各種形質を測定した。 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]
Genomic DNA was extracted from wild rice Oryza nivara with A genome, and BAC library was created. From the library, BAC clones with sh4 gene region genomic fragments were designed with specific primers to increase only the sh4 region by PCR, and BAC clones with sh4 gene region were isolated with or without amplification by PCR, Then, after subcloning the DNA obtained by fragmenting the BAC clone DNA to a few kbp into the pUC18 vector, the end reading DNA sequence of each subclone was determined and assembled, so that the sh4 gene genomic region of O.nivara The DNA sequence was determined. From the obtained genomic fragment sequence information of the sh4 region, the promoter region is predicted from the information of the known sh4 gene product, and the sh4 gene region, about 8.8 kbp length is excised by digestion reaction with KpnI and BamHI restriction enzyme, pPZP2H The genomic fragment was introduced into the -lac vector to create a transformation construct. Then, an approximately 8.8 kb genomic fragment (FIG. 1, SEQ ID NO: 1) containing the coding region and the regulatory region of the isolated functional allele sh4 gene was transformed into two rice lines, Nipponbare, NIL (qSH1) (Konishi et al In 2006, the gene was introduced using the ultra-rapid transformation method of monocotyledons (Japanese Patent No. 314084) by the rice transformation method. Antibiotic hygromycin was used for selection of transformation. About 10 independent transformants were prepared, and various traits such as weight and shedding were measured.
〔実施例２〕
完熟したイネ種子を収穫し、各形質転換系統から、稔実粒を5個ずつ、個体ごとに選び、その質量を測定した。ポジション効果によると推定される形質変化に幅はあるもののベクターコントロールと比較した際に、有意な籾重の増加が確認でき、系統によっては、約１．５倍に増加した（図２）。外観上も籾のサイズの増加、玄米サイズの増加が確認できた（図３）。また、一穂粒数への影響は、ベクターコントロールの触れと同じで、sh4系統での変化はほとんど確認できなかった（図４）。 [Example 2]
Ripe rice seeds were harvested, and 5 seed grains were selected from each transformed line for each individual, and the mass was measured. Although there was a range of phenotypic changes presumed to be due to the position effect, a significant increase in weight was confirmed when compared with the vector control, which increased about 1.5 times depending on the line (FIG. 2). In terms of appearance, an increase in the size of the koji and an increase in the size of the brown rice were confirmed (FIG. 3). In addition, the effect on the number of spikelets was the same as that of the vector control, and almost no change was observed in the sh4 line (FIG. 4).
完熟したイネ種子を収穫し、各形質転換系統から、稔実粒を5個ずつ、個体ごとに選び、その質量を測定した。ポジション効果によると推定される形質変化に幅はあるもののベクターコントロールと比較した際に、有意な籾重の増加が確認でき、系統によっては、約１．５倍に増加した（図２）。外観上も籾のサイズの増加、玄米サイズの増加が確認できた（図３）。また、一穂粒数への影響は、ベクターコントロールの触れと同じで、sh4系統での変化はほとんど確認できなかった（図４）。 [Example 2]
Ripe rice seeds were harvested, and 5 seed grains were selected from each transformed line for each individual, and the mass was measured. Although there was a range of phenotypic changes presumed to be due to the position effect, a significant increase in weight was confirmed when compared with the vector control, which increased about 1.5 times depending on the line (FIG. 2). In terms of appearance, an increase in the size of the koji and an increase in the size of the brown rice were confirmed (FIG. 3). In addition, the effect on the number of spikelets was the same as that of the vector control, and almost no change was observed in the sh4 line (FIG. 4).
〔実施例３〕
機能型sh4を導入した系統で、玄米サイズの増加が顕著な個体の自殖後代Ｔ１系統に付いた穂を観察した。Ｔ０と同等な籾サイズの増加が確認できた（図５）。 Example 3
In the line in which the functional type sh4 was introduced, the ears attached to the T1 line of progeny progeny of individuals whose brown rice size was significantly increased were observed. An increase in the cocoon size equivalent to T0 was confirmed (FIG. 5).
機能型sh4を導入した系統で、玄米サイズの増加が顕著な個体の自殖後代Ｔ１系統に付いた穂を観察した。Ｔ０と同等な籾サイズの増加が確認できた（図５）。 Example 3
In the line in which the functional type sh4 was introduced, the ears attached to the T1 line of progeny progeny of individuals whose brown rice size was significantly increased were observed. An increase in the cocoon size equivalent to T0 was confirmed (FIG. 5).
〔実施例４〕
実施例１に記載の方法により、品種日本晴にsh4遺伝子を導入した系統の自殖T1後代４個体について、人工気象室にてイネ植物体の栽培を行い、穂ごとのモミ重（mｇ）を測定した。
その結果、ベクターコントロール（ベクターのみを日本晴に導入した系統）及び日本晴と比較して、モミ重が有意に増加していることが明らかとなった（図７）。
また、これらの個体について、個体ごとの総籾数(稔実粒、不稔粒）（図８）、及び個体ごとの穂重（図９）を測定し、ベクターコントロール及び日本晴と比較した。
その結果、TO-3の後代は、ベクターコントロール及び日本晴と比較して、2倍強の収量性を示すことが明らかとなった（図８、図９）。一方、TO-5の後代は個体の稔実粒数・総籾数が少なく収量への効果はないことが明らかとなった（図８、図９）。 Example 4
According to the method described in Example 1, rice plants were cultivated in an artificial weather chamber for four self-breeding T1 progenies of the line that introduced the sh4 gene into the variety Nipponbare, and the fir weight (mg) per ear was measured. did.
As a result, it was revealed that the fir weight was significantly increased as compared with vector control (a line in which only a vector was introduced into Nipponbare) and Nipponbare (FIG. 7).
In addition, for these individuals, the total number of pods (fruit seeds, sterile grains) (FIG. 8) and panicle weight (FIG. 9) for each individual were measured and compared with Vector Control and Nipponbare.
As a result, it became clear that the progeny of TO-3 showed a yield of slightly more than twice that of Vector Control and Nipponbare (FIGS. 8 and 9). On the other hand, it became clear that the progeny of TO-5 has no effect on the yield because the number of seed berries and total number of potatoes is small (FIGS. 8 and 9).
実施例１に記載の方法により、品種日本晴にsh4遺伝子を導入した系統の自殖T1後代４個体について、人工気象室にてイネ植物体の栽培を行い、穂ごとのモミ重（mｇ）を測定した。
その結果、ベクターコントロール（ベクターのみを日本晴に導入した系統）及び日本晴と比較して、モミ重が有意に増加していることが明らかとなった（図７）。
また、これらの個体について、個体ごとの総籾数(稔実粒、不稔粒）（図８）、及び個体ごとの穂重（図９）を測定し、ベクターコントロール及び日本晴と比較した。
その結果、TO-3の後代は、ベクターコントロール及び日本晴と比較して、2倍強の収量性を示すことが明らかとなった（図８、図９）。一方、TO-5の後代は個体の稔実粒数・総籾数が少なく収量への効果はないことが明らかとなった（図８、図９）。 Example 4
According to the method described in Example 1, rice plants were cultivated in an artificial weather chamber for four self-breeding T1 progenies of the line that introduced the sh4 gene into the variety Nipponbare, and the fir weight (mg) per ear was measured. did.
As a result, it was revealed that the fir weight was significantly increased as compared with vector control (a line in which only a vector was introduced into Nipponbare) and Nipponbare (FIG. 7).
In addition, for these individuals, the total number of pods (fruit seeds, sterile grains) (FIG. 8) and panicle weight (FIG. 9) for each individual were measured and compared with Vector Control and Nipponbare.
As a result, it became clear that the progeny of TO-3 showed a yield of slightly more than twice that of Vector Control and Nipponbare (FIGS. 8 and 9). On the other hand, it became clear that the progeny of TO-5 has no effect on the yield because the number of seed berries and total number of potatoes is small (FIGS. 8 and 9).
〔実施例５〕
実施例１と同様の方法により、転流が良いという特徴を有する品種「にこまる」、及びイネの粒が大粒であるという特徴を有する品種「オオチカラ（大力）」に、sh4遺伝子を導入した。そして、これらの品種の形質転換当代T0個体についた稔実粒の平均モ ミ重(mg)を測定した。
その結果、「にこまる」においてもsh4遺伝子を導入することによって、日本晴と同様にイネの粒が重くなることが確認された（図１０）。また、オオチカラはもともと大粒であり一粒の重量が30mgであったが、sh4遺伝子を導入することによってその重量が40mgを超える形質転換系統を得られることが明らかとなった（図１１）。 Example 5
In the same manner as in Example 1, the sh4 gene was introduced into the cultivar “Nikomaru”, which has the characteristics of good commutation, and the cultivar “Ochikara (large power)”, which has the characteristics that the grain of rice is large. Then, the average fir weight (mg) of the persimmon grains per transgenic T0 individual of these varieties was measured.
As a result, it was confirmed that by introducing the sh4 gene in “Nikomaru”, rice grains become heavier as in Nipponbare (FIG. 10). In addition, it was clarified that the Ochikara was originally a large grain and the weight of one grain was 30 mg. However, by introducing the sh4 gene, a transformed line exceeding 40 mg was obtained (FIG. 11).
実施例１と同様の方法により、転流が良いという特徴を有する品種「にこまる」、及びイネの粒が大粒であるという特徴を有する品種「オオチカラ（大力）」に、sh4遺伝子を導入した。そして、これらの品種の形質転換当代T0個体についた稔実粒の平均モ ミ重(mg)を測定した。
その結果、「にこまる」においてもsh4遺伝子を導入することによって、日本晴と同様にイネの粒が重くなることが確認された（図１０）。また、オオチカラはもともと大粒であり一粒の重量が30mgであったが、sh4遺伝子を導入することによってその重量が40mgを超える形質転換系統を得られることが明らかとなった（図１１）。 Example 5
In the same manner as in Example 1, the sh4 gene was introduced into the cultivar “Nikomaru”, which has the characteristics of good commutation, and the cultivar “Ochikara (large power)”, which has the characteristics that the grain of rice is large. Then, the average fir weight (mg) of the persimmon grains per transgenic T0 individual of these varieties was measured.
As a result, it was confirmed that by introducing the sh4 gene in “Nikomaru”, rice grains become heavier as in Nipponbare (FIG. 10). In addition, it was clarified that the Ochikara was originally a large grain and the weight of one grain was 30 mg. However, by introducing the sh4 gene, a transformed line exceeding 40 mg was obtained (FIG. 11).
現在の世界の人口増加率と、穀物の生産量の伸びから推定すると、１０年後には深刻な穀物不足が国際的に起こると考えられる。また、バイオエネルギー生産による食料とエネルギー生産の競争激化により、穀物不足は早まる可能性がある。そこで、主要穀物であるイネの収量性を改善する本発明は、産業上大きな効果を示すものと考えられる。現在の世界人口の約半分、つまり、３０億人がコメを主食にしており、単純計算で、１％の増収は３０００万人の主食の確保に相当する。
If we estimate from the world population growth rate and the growth of grain production, a serious grain shortage will occur internationally in 10 years. Grain shortages may also be accelerated by intensifying competition between food and energy production through bioenergy production. Therefore, it is considered that the present invention for improving the yield of rice, which is a main grain, has a large industrial effect. About half of the current world population, that is, 3 billion people use rice as their staple food. By simple calculations, a 1% increase in sales is equivalent to securing 30 million staple foods.
Claims (13)
- 下記（ａ）から（ｄ）のいずれかに記載のDNAを含む、植物体の穀粒の大きさを増大させた植物体。
（ａ）配列番号：３に記載のアミノ酸配列からなるタンパク質をコードするDNA
（ｂ）配列番号：１又は２に記載の塩基配列のコード領域を含むDNA
（ｃ）配列番号：３に記載のアミノ酸配列において１または複数のアミノ酸が置換、欠失、付加、および／または挿入されたアミノ酸配列からなるタンパク質をコードするDNA
（ｄ）配列番号：１又は２に記載の塩基配列からなるDNAとストリンジェントな条件下でハイブリダイズするDNA The plant body which increased the size of the grain of the plant body containing DNA in any one of the following (a) to (d).
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 3
(B) DNA containing the coding region of the base sequence described in SEQ ID NO: 1 or 2
(C) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 3
(D) DNA that hybridizes under stringent conditions with DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1 or 2 - 前記植物体が単子葉植物である、請求項１に記載の植物体。 The plant body according to claim 1, wherein the plant body is a monocotyledonous plant.
- 植物体がイネ科植物である、請求項１に記載の植物体。 The plant body according to claim 1, wherein the plant body is a gramineous plant.
- 下記（ａ）から（ｄ）のいずれかに記載のDNAが発現するように導入されたベクター。
（ａ）配列番号：３に記載のアミノ酸配列からなるタンパク質をコードするDNA
（ｂ）配列番号：１又は２に記載の塩基配列のコード領域を含むDNA
（ｃ）配列番号：３に記載のアミノ酸配列において１または複数のアミノ酸が置換、欠失、付加、および／または挿入されたアミノ酸配列からなるタンパク質をコードするDNA
（ｄ）配列番号：１又は２に記載の塩基配列からなるDNAとストリンジェントな条件下でハイブリダイズするDNA A vector introduced so that the DNA according to any one of (a) to (d) below is expressed.
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 3
(B) DNA containing the coding region of the base sequence described in SEQ ID NO: 1 or 2
(C) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 3
(D) DNA that hybridizes under stringent conditions with DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1 or 2 - 請求項４に記載のベクターが導入された宿主細胞。 A host cell into which the vector according to claim 4 has been introduced.
- 請求項４に記載のベクターが導入された植物細胞。 A plant cell into which the vector according to claim 4 is introduced.
- 請求項６に記載の植物細胞を含む形質転換植物体。 A transformed plant comprising the plant cell according to claim 6.
- 請求項７に記載の形質転換植物体の子孫またはクローンである、形質転換植物体。 A transformed plant that is a descendant or clone of the transformed plant according to claim 7.
- 請求項７または８に記載の形質転換植物体の繁殖材料。 The propagation material of the transformed plant body of Claim 7 or 8.
- 下記（ａ）から（ｄ）のいずれかに記載のDNAを植物体の細胞内で発現させる工程を含む、植物体の穀粒の大きさを増大させる方法。
（ａ）配列番号：３に記載のアミノ酸配列からなるタンパク質をコードするDNA
（ｂ）配列番号：１又は２に記載の塩基配列のコード領域を含むDNA
（ｃ）配列番号：３に記載のアミノ酸配列において１または複数のアミノ酸が置換、欠失、付加、および／または挿入されたアミノ酸配列からなるタンパク質をコードするDNA
（ｄ）配列番号：１又は２に記載の塩基配列からなるDNAとストリンジェントな条件下でハイブリダイズするDNA The method to increase the grain size of a plant body including the process of expressing the DNA in any one of following (a) to (d) in the cell of a plant body.
(A) DNA encoding a protein comprising the amino acid sequence set forth in SEQ ID NO: 3
(B) DNA containing the coding region of the base sequence described in SEQ ID NO: 1 or 2
(C) DNA encoding a protein comprising an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted in the amino acid sequence of SEQ ID NO: 3
(D) DNA that hybridizes under stringent conditions with DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1 or 2 - 前記植物体が単子葉植物である、請求項１０に記載の方法。 The method according to claim 10, wherein the plant body is a monocotyledonous plant.
- 前記植物体がイネ科植物である、請求項１０に記載の方法。 The method according to claim 10, wherein the plant body is a gramineous plant.
- 交配により、前記DNAを植物体に導入することを特徴とする、請求項１０～１２のいずれかに記載の方法。 The method according to any one of claims 10 to 12, wherein the DNA is introduced into a plant body by crossing.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009801295337A CN102112610B (en) | 2008-05-29 | 2009-05-29 | Plant having increased grain size which contains Sh4 gene |
| JP2010514547A JP5610440B2 (en) | 2008-05-29 | 2009-05-29 | A plant body containing the sh4 gene and having an increased grain size of the plant body |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2008-141357 | 2008-05-29 | ||
| JP2008141357 | 2008-05-29 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2009145290A1 true WO2009145290A1 (en) | 2009-12-03 |
Family
ID=41377159
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/JP2009/059849 WO2009145290A1 (en) | 2008-05-29 | 2009-05-29 | Plant having increased grain size which contains sh4 gene |
Country Status (4)
| Country | Link |
|---|---|
| JP (1) | JP5610440B2 (en) |
| KR (1) | KR20110010816A (en) |
| CN (1) | CN102112610B (en) |
| WO (1) | WO2009145290A1 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8722072B2 (en) | 2010-01-22 | 2014-05-13 | Bayer Intellectual Property Gmbh | Acaricidal and/or insecticidal active ingredient combinations |
| US9265252B2 (en) | 2011-08-10 | 2016-02-23 | Bayer Intellectual Property Gmbh | Active compound combinations comprising specific tetramic acid derivatives |
| JP2019126339A (en) * | 2018-01-23 | 2019-08-01 | 国立研究開発法人理化学研究所 | Production method of plant whose seed is increased in size |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1817900A (en) * | 2006-03-15 | 2006-08-16 | 中国农业大学 | Transcription factor for regulating plant fallen, its coding gene and use |
-
2009
- 2009-05-29 CN CN2009801295337A patent/CN102112610B/en not_active Expired - Fee Related
- 2009-05-29 WO PCT/JP2009/059849 patent/WO2009145290A1/en active Application Filing
- 2009-05-29 KR KR1020107029058A patent/KR20110010816A/en not_active Application Discontinuation
- 2009-05-29 JP JP2010514547A patent/JP5610440B2/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1817900A (en) * | 2006-03-15 | 2006-08-16 | 中国农业大学 | Transcription factor for regulating plant fallen, its coding gene and use |
Non-Patent Citations (1)
| Title |
|---|
| CHANGBAO LI ET AL.: "Rice Domestication by Reducing Shattering", SCIENCE, vol. 311, 2006, pages 1936 - 1939 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8722072B2 (en) | 2010-01-22 | 2014-05-13 | Bayer Intellectual Property Gmbh | Acaricidal and/or insecticidal active ingredient combinations |
| US9265252B2 (en) | 2011-08-10 | 2016-02-23 | Bayer Intellectual Property Gmbh | Active compound combinations comprising specific tetramic acid derivatives |
| JP2019126339A (en) * | 2018-01-23 | 2019-08-01 | 国立研究開発法人理化学研究所 | Production method of plant whose seed is increased in size |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102112610B (en) | 2013-12-18 |
| KR20110010816A (en) | 2011-02-07 |
| CN102112610A (en) | 2011-06-29 |
| JP5610440B2 (en) | 2014-10-22 |
| JPWO2009145290A1 (en) | 2011-10-13 |
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