WO2009145290A1 - Plante ayant une dimension de grain accrue et contenant le gène sh4 - Google Patents

Plante ayant une dimension de grain accrue et contenant le gène sh4 Download PDF

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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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plant
dna
seq
gene
amino acid
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PCT/JP2009/059849
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English (en)
Japanese (ja)
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毅 井澤
左江子 杉田
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独立行政法人農業生物資源研究所
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Priority to CN2009801295337A priority Critical patent/CN102112610B/zh
Priority to JP2010514547A priority patent/JP5610440B2/ja
Publication of WO2009145290A1 publication Critical patent/WO2009145290A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/823—Reproductive tissue-specific promoters
    • C12N15/8234—Seed-specific, e.g. embryo, endosperm
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

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).

Abstract

L'invention porte sur une plante qui présente une dimension de grain accrue et qui contient le gène sh4 de type fonctionnel. Des études exhaustives ont été faites sur les gènes intervenant dans les dimensions de grain de plantes, et il s'est avéré qu'une nouvelle fonction consistant à augmenter les dimensions de balles de riz, à favoriser la  translocation et, par conséquent, à augmenter les dimensions des grains, peut être exprimée dans un cultivar de plants de riz (« Nihonbare », « Nikomaru », « Oochikara ») par l'introduction d'un allèle fonctionnel du gène sh4 issu d'un plant de riz de type sauvage dans le cultivar de plant de riz par transformation.
PCT/JP2009/059849 2008-05-29 2009-05-29 Plante ayant une dimension de grain accrue et contenant le gène sh4 WO2009145290A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN2009801295337A CN102112610B (zh) 2008-05-29 2009-05-29 含有Sh4基因的具有增加的颗粒大小的植物
JP2010514547A JP5610440B2 (ja) 2008-05-29 2009-05-29 ｓｈ４遺伝子を含む、植物体の穀粒サイズを増大させた植物体

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JP2008141357 2008-05-29
JP2008-141357 2008-05-29

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Cited By (3)

* Cited by examiner, † Cited by third party
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 (ja) * 2018-01-23 2019-08-01 国立研究開発法人理化学研究所 種子のサイズが増大した植物の製造方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1817900A (zh) * 2006-03-15 2006-08-16 中国农业大学 一种调控植物落粒性的转录因子及其编码基因与应用

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1817900A (zh) * 2006-03-15 2006-08-16 中国农业大学 一种调控植物落粒性的转录因子及其编码基因与应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
CHANGBAO LI ET AL.: "Rice Domestication by Reducing Shattering", SCIENCE, vol. 311, 2006, pages 1936 - 1939 *

Cited By (3)

* Cited by examiner, † Cited by third party
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 (ja) * 2018-01-23 2019-08-01 国立研究開発法人理化学研究所 種子のサイズが増大した植物の製造方法

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CN102112610A (zh) 2011-06-29
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JP5610440B2 (ja) 2014-10-22
JPWO2009145290A1 (ja) 2011-10-13

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