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WO2010093015A1 - Gène associé à la tolérance à l'eau profonde et son utilisation - Google Patents

Gène associé à la tolérance à l'eau profonde et son utilisation Download PDF

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WO2010093015A1
WO2010093015A1 PCT/JP2010/052074 JP2010052074W WO2010093015A1 WO 2010093015 A1 WO2010093015 A1 WO 2010093015A1 JP 2010052074 W JP2010052074 W JP 2010052074W WO 2010093015 A1 WO2010093015 A1 WO 2010093015A1
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plant
polynucleotide
seq
protein
gene
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PCT/JP2010/052074
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English (en)
Japanese (ja)
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基行 芦苅
洋子 服部
隆 松本
建忠 呉
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国立大学法人名古屋大学
独立行政法人農業生物資源研究所
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Publication of WO2010093015A1 publication Critical patent/WO2010093015A1/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
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G22/00—Cultivation of specific crops or plants not otherwise provided for
    • A01G22/20—Cereals
    • A01G22/22—Rice
    • 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

Definitions

  • the present invention relates to isolation and identification of a gene relating to a response (flooding response) to an increase in water depth of a plant, and a method for improving the flooding tolerance of a plant using the gene.
  • Rice (0ryza sativa) is considered to be a semi-aqueous plant, depending on the need for water and the system for water, upland rice (lowland rice) and floating rice (deep water rice) (deepwater rice or floating rice) Can be classified.
  • Land rice and water rice are cultivated in high altitudes or shallow water areas (about 10 cm deep), but when the water level rises due to the rainy season or heavy rain in these cultivated areas, rice is submerged, and this type of rice dies due to oxygen deficiency. Resulting in.
  • floating rice can take a strategy of avoiding flooding by extending the internodes of the stem according to the water level. Floating rice is able to survive against flooding by increasing water depth, i.e.
  • Non-Patent Document 1 Such dramatic and rapid growth in response to flooding is one of the unique biological adaptations to harsh environments.
  • Non-Patent Documents 2 and 3 Molecular genetic approaches such as cDNA and microarrays have been carried out to elucidate the mechanism for flood response. Moreover, some genes involved in the submergence response have been clarified by classical experiments on the submergence response (Non-Patent Documents 2 and 3). In addition, physiological studies on floating rice suggest that ethylene, abscisic acid and gibberellin are involved in the flooding response (Non-Patent Document 4).
  • An object of the present invention is to isolate and identify a gene related to the submergence response and to use the gene.
  • the present inventors tried to identify genes involved in QTL analysis combined with positional cloning in order to isolate and identify the genes involved in the submergence response in rice and to understand the molecular mechanism of submergence response. .
  • the present inventors succeeded in isolating and identifying a submergence response-related gene for the first time, and further introducing the identified gene into a non-floating rice species to obtain a submergence response capability.
  • a converted plant was obtained. According to the present invention, the following means are provided based on these findings.
  • the polynucleotide of the present invention may be derived from rice.
  • B a protein having an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence represented by SEQ ID NO: 4, and having internode elongation activity of stems during submergence Encoding polynucleotide.
  • (C) A polynucleotide encoding a protein having an amino acid sequence having 70% or more identity with the amino acid sequence represented by SEQ ID NO: 4 and having stem internode elongation activity during submergence.
  • (D) A polynucleotide comprising the base sequence represented by SEQ ID NO: 3.
  • (E) a polynucleotide that hybridizes with a complementary strand of a polynucleotide comprising the base sequence represented by SEQ ID NO: 3 under stringent conditions and encodes a protein having internode elongation activity of a stem when submerged.
  • (F) A polynucleotide encoding a protein having 70% or more identity with the base sequence represented by SEQ ID NO: 3 and having internode elongation activity of stems during submergence.
  • a vector comprising the polynucleotide, a plant cell into which the vector has been introduced, a transformed plant comprising the plant cell, a progeny or clone of the plant, and a propagation material for the plant.
  • a method for producing a transformed plant comprising the steps of introducing the polynucleotide of the present invention into a plant cell and regenerating the plant from the plant cell.
  • the cultivation step may include a step S that induces a hand completion response in the transformed plant body.
  • FIG. 2 shows sequence specific binding of OsEIN3 protein to SK1 and SK2. It is a figure which shows the competition test result by the mutant of the promoter region of SK1 and SK2. It is a figure which shows the submergence treatment time and the density
  • the present invention relates to genes involved in submergence response and their use.
  • the present invention is based on the fact that the present inventors succeeded in isolating and identifying a gene that triggers a submergence response for the first time as follows.
  • NIL-12 near-isogenic line
  • NIL12 hetero-isogenic line 16000 derived from T65 / C9285 hybrids (FIG. 2) and NIL- derived from T65 / Bhadua hybrids.
  • a 12B hetero near-isogenic line 12000 strain (FIG. 2) was prepared, and the QTL was subjected to positional cloning.
  • Candidate regions for QTL are duplicated in two independent populations.
  • a BAC library covering this duplicated candidate region was further constructed.
  • additional regions were added in C9285. A region was found. The total 67.5 kb DNA fragment found was predicted to have 18 ORFs (FIG. 5).
  • SK1 and SK2 were not expressed in the air, but were strongly expressed in deep water conditions (FIGS. 6 and 7). Therefore, in order to confirm that SK1 and SK2 induce a submergence response, three subclones carrying one or both of the two genes (SK1, SK2) are selected from the sub-library of the BAC library. These were used to transform T65, a non-floating rice species, using these. Similarly, a BAC library was prepared for Bhadua species, and T65 was transformed with SK1 and SK2.
  • a transformed plant having flood resistance or improved can be obtained by modifying a plant using a gene newly isolated and identified by the present inventors.
  • the genes found by the present inventors are particularly useful in the agricultural field, the energy field using biomass as a raw material, and the chemical industry field. For example, it is possible to obtain plants such as rice that can survive and be harvested against flooding during the rainy season or during heavy rain. Moreover, since a plant with stems and leaves extended is obtained, a plant with a high yield can be obtained when the stems and leaves are used as useful parts (for food or as biomass).
  • the gene isolated by the present inventors When a plant is created using a gene isolated and identified by the present inventors, it is preferable to use transformation.
  • the period required for transformation is extremely short compared to gene transfer by mating, and it is possible to impart or improve submergence tolerance without changing other traits.
  • the flood resistance can be easily improved.
  • the gene isolated by the present inventors can be expected to be applied to grain breeding such as wheat, barley and corn.
  • the gene isolated by the present inventors is an ethylene-responsive transcription factor and widely distributed in plants, it is considered that introduction of the gene can improve the water-resistant response in all plants.
  • genes found by the present inventors are considered to be used for plants including other gramineous plants (for example, wheat, barley, corn, sugarcane, sorghum, etc.) other than rice. Useful in the chemical industry.
  • the polynucleotide of the present invention encodes a protein that is a nuclear localization ethylene-responsive transcription factor and induces internode elongation of stems as a submergence response (the protein of the present invention, hereinafter also referred to as the present protein).
  • the present protein will be described.
  • the protein has a nuclear localization signal (NLS).
  • the nuclear localization signal is usually composed mainly of basic amino acids.
  • Examples of the NLS of this protein include, but are not limited to, SEQ ID NO: 5 and SEQ ID NO: 6 (both are deduced sequences).
  • this protein can have an ERF domain which is a DNA binding domain. According to the phylogenetic analysis of certain ERF domains (AP2 / ERF domains) (SEQ ID NO: 7 and SEQ ID NO: 8) encompassed by this protein, these amino acid sequences are similar to OsSUB1 protein and OsERF I know that.
  • SEQ ID Nos: 5 and 7 are NLS (presumed) and ERF in the amino acid sequence represented by SEQ ID NO: 2
  • SEQ ID Nos: 6 and 8 are NLS (presumed) and NLS in the amino acid sequence represented by SEQ ID NO: 4. ERF.
  • This protein can have ethylene-responsive transcription factor activity on the C-terminal side. More specifically, it preferably has ethylene-responsive transcription factor activity on the C-terminal side of the ERF domain retained by the protein.
  • the ethylene-responsive transcription factor activity can be detected, for example, by the method disclosed in the examples in the subsequent stage.
  • proteins having the amino acid sequences represented by SEQ ID NO: 2 and SEQ ID NO: 4. These proteins were isolated from C9285, a floating rice cultivar native to Bangladesh.
  • a protein having an amino acid sequence highly identical to the amino acid sequence represented by SEQ ID NO: 2 or 4, or 1 or 2 in the amino acid sequence represented by SEQ ID NO: 2 or 4 A protein having an amino acid sequence in which a plurality of amino acids are substituted, deleted, added, and / or inserted, and is encoded by a base sequence that hybridizes under stringent conditions to a complementary strand of the base sequence represented by SEQ ID NO: 1 or 3. And a protein having an amino acid sequence.
  • Examples of the present polynucleotide include a polynucleotide encoding the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4. Each of these polynucleotides can be used alone. In that case, a polynucleotide encoding the amino acid sequence represented by SEQ ID NO: 4 can be preferably used. It is more preferable to use both of these as the present polynucleotide.
  • Examples of the polynucleotide encoding the amino acid sequence represented by SEQ ID NO: 2 and SEQ ID NO: 4 include polynucleotides having the base sequences represented by SEQ ID NO: 1 and SEQ ID NO: 3, respectively. Such polynucleotides can be prepared by known techniques.
  • total mRNA is prepared from a rice tissue extract, a primer is designed based on the nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3, and PCR method such as RACE method is performed to perform the full length of the above SEQ ID NO: cDNA can be obtained.
  • a cDNA library can be prepared from a rice tissue extract, a probe can be designed based on the above base sequence, and obtained by a hybridization method or the like. Further, it may be artificially synthesized based on the base sequence described in the above SEQ ID NO.
  • This polynucleotide is not limited to those encoding the amino acid sequences represented by SEQ ID NOs: 2 and 4, but encodes a protein exhibiting an activity of extending between stem nodes during submergence (submergence responsive activity) If it is.
  • a polynucleotide may be prepared from nature or artificially prepared as long as it has the above-mentioned submergence responsive activity. For example, orthologs, homologues, and artificially introduced mutations of the above SEQ ID NO.
  • the submergence responsive activity can be evaluated by at least one of the methods disclosed in the Examples.
  • a transformed plant can be prepared and measured by measuring internode elongation of the plant during submergence or by measuring internode elongation of the plant by exposure to ethylene or ethephon in the air.
  • This polynucleotide has a high degree of identity with these sequences by performing a homology search against the plant genome database using the amino acid sequence of SEQ ID NO: 2 or 4 or the base sequence of SEQ ID NOs: 1, 3. It may be. For example, based on information on the amino acid sequences of NLS and ERF or the base sequences encoding them from grasses or other plants such as wild and cultivated varieties such as rice that exhibit internode elongation of stems as a submergence response In addition, it may encode an amino acid sequence extracted by homology search using a plant genome database such as a rice genome database.
  • the polynucleotide preferably encodes an amino acid sequence having high identity with the amino acid sequence represented by SEQ ID NO: 2 or 4, and high identity is at least 40% or more, preferably 60% or more, and more preferably Means 80% or more, more preferably 90% or more, more preferably at least 95% or more, more preferably at least 97% or more (eg, 98 to 99%) of sequence identity.
  • the identity of the amino acid sequence and the base sequence is, for example, the algorithm BLAST (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990, Proc. Natl. Acad. Sci. USA 90: 5873-5877 by Karlin and Altschul. , 1993).
  • the present polynucleotide may encode an amino acid sequence in which one or more amino acids are substituted, deleted, added, and / or inserted in the amino acid sequence represented by SEQ ID NO: 2 or 4. It can be prepared by introducing a mutation site-specifically or randomly by applying a gene modification method known to those skilled in the art, such as a mutation introduction method by PCR or a cassette mutation method. Alternatively, it is also possible to synthesize a sequence in which a mutation is introduced into the base sequence described in the above SEQ ID No. using a commercially available nucleic acid synthesizer.
  • the present polynucleotide may be one that hybridizes under stringent conditions to a complementary strand of the base sequence represented by SEQ ID NO: 1 or 3.
  • stringent hybridization conditions can be appropriately selected by those skilled in the art. For example, 25% formamide, 50% formamide under more severe conditions, 4 ⁇ SSC, 50 mM HEPES® (pH 7.0), 10 ⁇ Denhardt's solution, hybridization solution containing 20 ⁇ g / ml denatured salmon sperm DNA at 42 ° C. After overnight prehybridization, add a labeled probe and incubate at 42 ° C. overnight.
  • the cleaning solution and temperature conditions for the subsequent cleaning are about ⁇ 1xSSC, 0.1% SDS, 37 ° C '', more severe conditions are about ⁇ 0.5xSSC, 0.1% SDS, 42 ° C '', and more severe conditions are ⁇ 0.2xSSC , 0.1% SDS, 65 ° C. ”.
  • isolation of a polynucleotide having high homology with the probe sequence can be expected as the conditions for washing for hybridization become more severe.
  • combinations of the above SSC, SDS, and temperature conditions are exemplary, and those skilled in the art will understand the above or other factors that determine the stringency of hybridization (eg, probe concentration, probe length, hybridization reaction).
  • Such a polynucleotide is usually at least 40% or more, preferably 60% or more, more preferably 80% or more, more preferably 90% or more, more preferably at least 95% with the base sequence represented by SEQ ID NO: 1 or 3. More preferably, the sequence identity is at least 97% or more (for example, 98 to 99%).
  • the present polynucleotide includes genomic DNA, cDNA, and chemically synthesized DNA, and may be a DNA / RNA hybrid or a DNA / RNA chimera.
  • the base of RNA may be provided, and the base and sugar chain which chemically modified the natural base may be provided.
  • the polynucleotide does not ask its origin.
  • the polynucleotide is preferably derived from a plant. As described in the Examples, the present polynucleotide is isolated from rice (Poaceae), but it is considered to exist as long as it exhibits a similar flooding response.
  • the present polynucleotide is preferably derived from a monocotyledonous plant, more preferably from a grass family.
  • the expression vector of the present invention is an expression vector that retains the present polynucleotide.
  • the vector of the present invention is used as a vector for expressing the present polynucleotide in plant cells in order to produce the present protein and also to produce transformed plants.
  • 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 containing a polyadenylation site necessary for stabilization of the transcript.
  • the vector used for the transformation of plant cells is not particularly limited as long as the inserted gene can be expressed in the cells.
  • plant cells include 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.
  • promoters for constant expression include cauliflower mosaic virus 35S promoter (Odell et al. 1985 Nature 313: 810), rice actin promoter (Zhang et al.1991 Plant Cell 3: 1155), maize Ubiquitin promoter (Cornejo et al. 1993 Plant Mol.Biol. 23: 567) and the like.
  • promoters for inducible expression are known to be expressed by external factors such as infection and invasion of filamentous fungi / bacteria / viruses, low temperature, high temperature, drying, ultraviolet irradiation, spraying of specific compounds, etc. Promoters and the like. Examples of such promoters include rice chitinase gene promoter (Xu et al. 1996 Plant Mol. Biol. 30: 387) and tobacco PR protein gene promoter (Ohshima et al. 1990 Plan Cell 2:95), Rice “lip19” promoter (Aguan et al. 1993 Mol.GenGenet. 240: 1), rice “hsp80” and “hsp72” promoters (Van Breusegem et al.
  • the transformed cell of the present invention is a 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, in addition to cells such as E. coli, yeast, animal and plant cells and insect cells used for production of recombinant proteins.
  • a plant cell For example, cells, such as Arabidopsis thaliana, rice, corn, potato, and 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.
  • 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.
  • the transformed plant of the present invention contains a plant cell into which the vector of the present invention has been introduced.
  • a transformed plant can be obtained by regenerating a plant from a plant cell transformed by introducing the vector of the present invention.
  • Transformed plant cells can be prepared by a known method as described above. For example, gene transfer into protoplasts using polyethylene glycol (Datta, SK (1995) In Gene Transfer To Plants (Potrykus I and Spangenberg Eds.) Pp66- 74), gene transfer to protoplasts by electric pulse (Toki et al. (1992) Plant Physiol. 100, 1503-1507), gene transfer directly to cells by particle gun method (Christou et al.
  • 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.
  • the plant body produced in this way has been given or improved the ability to respond to submergence, and has improved resistance to submergence. Therefore, it is a plant body that can be expected to be harvested even in submersion and can be expected to increase the yield of the foliage part.
  • the plant produced in this way is not only flooded artificially or exposed to ethylene in the air, intentionally generating a flooding response, resulting in stem nodes.
  • the space can be extended. For this reason, a plant body with a high plant height or a large number of stems and leaves can be easily obtained.
  • the method for producing useful crops of the present invention comprises a step of cultivating the transformed plant of the present invention and a step of harvesting the transformed plant.
  • a crop can survive even during a rainy season or flooding due to heavy rain, so that the crop can be reliably harvested.
  • the cultivation process may include the step of inducing a flooding response in the transformed plant body.
  • the submergence response can be induced artificially by flooding the transformant or exposing it to ethylene. By carrying out such a treatment, it is possible to induce a submergence response in the transformed plant body and extend the internodes of the stem. As a result, it becomes possible to harvest plants having a high plant height or a large number of foliage.
  • the water level is 50% or more of the height of the plant, more preferably 70% or more, still more preferably 80% or more, still more preferably 90% or more, most Preferably, it is enough to be completely submerged.
  • exposure to ethylene can be supplied as ethylene gas, for example, in a gas atmosphere where plants can survive, such as in the air. Appropriate ethylene concentration and exposure time in the gas atmosphere can be determined in advance.
  • Wild rice W0106 (O. rufipogon; one year) native to India and cultivated species Taichung 65 (T65; O. sativa ssp. Japonica) are used for rice that does not have floating rice characteristics (hereinafter, non-floating rice species or controls) Used for crossing). Furthermore, O. glumaepatula (W2199) was used as a cultivated species. These lines, C9285, W0120, W0106 and O. glumaepatula were all obtained from the National Institute of Genetics (Japan), and T65 was cultivated at Nagoya University.
  • Plants were germinated by treatment in water in a petri dish at 30 ° C. for 72 hours, and then transplanted to pots having a diameter of 10 cm and a height of 13 cm.
  • TIL internode elongation length
  • BAC libraries were constructed from young leaves of plants in accordance with a conventional method. That is, partial digestion of DNA with HindIII, size fractionation of large DNA by pulse field gel electrophoresis (CHEF, Bio-Rad Laboratories, Hercules, California, USA), vector (pIndigo BAC-5, EPICENTRE Biotechnologies, Madison, Wisconsin, USA) and transformation into E. coli (DH10B strain). Positive BAC clones were screened by PCR from the pooled DNA for each of a sufficient number of BAC libraries holding total DNA equivalent to at least 6 times the amount of rice genomic DNA. For these BAC libraries, positive clones were screened by Southern hybridization from a high-density BAC filter.
  • SK1 and SK2 phenotypes evaluation of SK1 and SK2 phenotypes (measurement of internode elongation length) under submerged conditions was performed using F3 and F4 individuals. Phenotypes were compared for DNA fragments within the candidate regions of C9285, Bhadua and T65.
  • the full length genomic DNAs of SK1 and SK2 including the promoter region were introduced into the binary vector pBI-Hm12. These DNA fragments were introduced into T65, which is a japonica species, by the Agrobacterium method. As a control, an empty vector without these DNA fragments was introduced into T65.
  • RNA isolation and semi-quantitative RT-PCR Total RNA was prepared by the method of Samblook et al. (Molecular Cloning A Laboratory Manual Cold Spring Harbor, 1989) and the first strand of cDNA was prepared using 2 ⁇ g of the Omniscript Reverse Transcription Kit (Qiagen, California, USA). Synthesized from total RNA. Semi-quantitative RT-PCR was performed by the method of Kaneko et al. (Kaneko et al., 2003) using gene specific primers.
  • SK1 and SK2 cDNAs were amplified and introduced into pCR4 Blunt-TOPO (Invitrogen, California, USA). did. Analysis of the base sequence of this plasmid confirmed that SK1 and SK2 were correctly introduced.
  • the plasmid was subjected to restriction enzyme treatment, and the DNA fragment was introduced into the pBI101 binary vector having ACTIN1 promoter and nos terminator. This binary vector was introduced into Agrobacterium strain EHA101 (A. tumefaciens strain EHA101 (Hood et al., 1986)) by electroporation.
  • This sample was used to stain the nucleus and evaluate the intracellular localization of SK1 and SK2 derivatives at 2 ⁇ g / ⁇ L of 4 ′, 6-diamidino-2-phenylindole n-hydrate (DAPI; Dojindo, Kumamoto , Japan) Immerse in the solution.
  • DAPI 6-diamidino-2-phenylindole n-hydrate
  • the stained sample was observed with a confocal microscanning laser microscope (FV500; Olympus, Tokyo, Japan).
  • Plant hormones (auxin (IAA), brassinosteroid (BR), cytokinin (CK), gibberellin (GA), abscisic acid (ABA)) are about 100 mg (raw weight) of rice stem. Extracted from. These plant hormones are prepared by liquid chromatography-mass spectroscopy system chromatography (UPLC / Quattro Premier XE; Waters, Massachusetts, USA) and ODS column (Acquity) according to the method described in the literature (Hirano et al. 2008). -UPLC BEH-C 18 , 1.7 ⁇ m, 2.1 x 100 mm, Waters). The ethylene content was measured using a gas chromatograph (Hitachi, 263-30). The stem base including nodes and internodes was harvested from the flooding after 1 day, placed in a 6 ml glass vial, and held for 1 hour. The amount of ethylene in 1 ml of gas collected from the vial was measured.
  • IAA auxin
  • BR brassinosteroid
  • Electrophoretic gel mobility shift assay To produce OsEIL1 protein, its full-length DNA (Mao et al. 2006) was amplified by PCR, and the pET-32a (+) vector (Novagen, Madison, Wisconsin, USA) Introduced to XbaI and BglII sites. Probes of the SK1 and SK2 promoter fragments were labeled by introducing [ 32 P] dATP Klenow fragment into the 5 ′ overhang site. The DNA binding reaction was performed for 30 minutes at 4 ° C.
  • Electrophoresis was performed on a 13% acrylamide gel at 200 V for 2 hours in glycerin, 1 mM EDTA, 0.05% NP-40, and 2 0.25 ⁇ Tris-borate-EDTA buffer.
  • the competition test was performed by adding an unlabeled competitive oligonucleotide to the binding reaction and then adding a labeled oligonucleotide.
  • Non-Patent Documents 2 and 3 The present inventors have already found three main QTLs present on chromosomes 1, 3, and 12, respectively, using a hybrid of T65 and C9298.
  • Non-Patent Documents 2 and 3 QTL on chromosome 12 is considered to be most involved in the water depth increasing response.
  • a near-isogenic line (NIL) having a genetic background of T65 and having C9285 and Bhadua gene fragments at the end of chromosome 12. -12 was produced by the above method.
  • NIL-12 responded to flooding with internodes and leaf elongation.
  • a C9285 BAC library was constructed according to the above method, and it was found that one BAC clone (C9285_10H05) covers the candidate region. The results are shown in FIG. From the analysis result of the base sequence of the BAC clone, an additional gene region of 46 kb was detected in C9285 but not in T65. The total 67.5 kb DNA fragment had 18 putative ORFs.
  • the coding region base sequences of the SK1 gene and SK2 gene are shown in SEQ ID NOs: 1 and 3, respectively, and the amino acid sequences of the proteins encoded by these genes are shown in SEQ ID NOs: 2 and 4, respectively.
  • the SK1 gene coding region and its base sequence containing at least part of the promoter region on its 5 ′ side are shown in SEQ ID NO: 9, and the SK2 gene coding region and its base containing at least part of the promoter region on its 5 ′ side.
  • the sequence is shown in SEQ ID NO: 10.
  • FIG. 7 shows the results of expression analysis for SK1 and SK2.
  • total RNA was extracted from C9285 and T65 submerged for a predetermined time (3 hours, 6 hours, 12 hours) as shown in FIG. 7, and RT-PCR was performed using specific primers for these genes. It was performed using. The actin gene was used as a control.
  • SK1 was particularly strongly expressed in the submerged condition among the two genes, Snorkel1 (SK1) and Snorkel2 (SK2).
  • a BAC library of Bhadua and selected clones covering the desired region was prepared, and it was also confirmed whether Bhadua has SK2 and SK1.
  • subclone a containing SK1
  • subclone b containing SK2
  • the transformed plant carrying SK1 hardly responded to submergence, whereas the transformed plant carrying SK2 showed significant internodal extension to submergence. It was. Transformed plants carrying SK1 and SK2 responded better than plants carrying either.
  • a transgenic plant having a T65 gene background and overexpressing SK1 or SK2 under the control of the actin promoter was prepared, and internode elongation in air was evaluated. The results are shown in FIG. As shown in FIG. 9, the control (introduced with the empty vector) did not respond in the air. However, in the case of an overexpressing SK1, the first to third internodes were elongated, and SK2 In the overexpressing body, the first to seventh internodes were elongated.
  • the ethylene concentration in the plant body was measured in air and under submerged conditions using a plant hormone quantification method. As shown in FIG. 16, it was found that the ethylene concentration of any plant body was about 2.5 times higher than that in air under flooding conditions. After submerging C9285 and T65 for a predetermined time (6 hours, 12 hours, 24 hours), total RNA was extracted, and expression analysis of ethylene gene (ACS) was performed. The results are shown in FIG. As shown in FIG. 17, it was found that ACS5, which converts SAM to ACC in the rate-limiting step of the ethylene synthesis pathway, was induced by both T65 and C9285 under submerged conditions. From the above, it was found that the synthesis of ethylene induced by activating the ethylene biosynthesis gene ACS5 under submerged conditions induces the expression of SK1 and SK2.
  • ethylene is synthesized by activating the expression of the ethylene biosynthetic enzyme ACS5 gene, and the accumulated ethylene induces the expression of ethylene-responsive transcription factors SK1 and SK2, thereby expressing the submergence response. Is to cause.
  • the GA concentration rises to such an extent that it induces internode elongation directly or indirectly.

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Abstract

L'invention porte sur l'isolement/l'identification d'un gène associé à la tolérance à l'eau profonde. L'invention porte également sur une utilisation du gène. Un polynucléotide codant pour une protéine comprenant la séquence d'acides aminés représentée dans SEQ ID NO:4 est isolé comme gène associé à la tolérance à l'eau profonde. L'utilisation du gène permet la production d'une plante qui peut présenter aisément une tolérance à l'eau profonde.
PCT/JP2010/052074 2009-02-13 2010-02-12 Gène associé à la tolérance à l'eau profonde et son utilisation WO2010093015A1 (fr)

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JP2009031885A JP5303713B2 (ja) 2009-02-13 2009-02-13 冠水応答関連遺伝子及びその利用
JP2009-031885 2009-02-13

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104885837A (zh) * 2015-07-03 2015-09-09 安徽袁粮水稻产业有限公司 一种北方地区直播稻耐淹性室内鉴定方法
CN104920089A (zh) * 2015-07-03 2015-09-23 宋立胜 一种直播稻育种耐淹亲本的筛选方法

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
HATTORI, Y. ET AL.: "A Major QTL Confers Rapid Internode Elongation in Response to Water Rise in Deepwater Rice", BREEDING SCIENCE, vol. 57, no. 4, 14 December 2007 (2007-12-14), pages 305 - 314 *
HATTORI, Y. ET AL.: "Mapping of three QTLs that regulate internode elongation in deepwater rice", BREEDING SCIENCE, vol. 58, no. 1, 26 March 2008 (2008-03-26), pages 39 - 46 *
HATTORI, Y. ET AL.: "The ethylene response factors SNORKEL1 and SNORKEL2 allow rice to adapt to deep water", NATURE, vol. 460, 20 August 2009 (2009-08-20), pages 1026 - 1030 *
YOKO HATTORI ET AL.: "Ukiine no Sekkan Shincho ni Kansuru QTL Kaiseki", DAI 47 KAI PROCEEDINGS OF THE ANNUAL MEETING OF THE JAPANESE SOCIETY OF PLANT PHYSIOLOGISTS, - 19 March 2006 (2006-03-19), pages 225 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104885837A (zh) * 2015-07-03 2015-09-09 安徽袁粮水稻产业有限公司 一种北方地区直播稻耐淹性室内鉴定方法
CN104920089A (zh) * 2015-07-03 2015-09-23 宋立胜 一种直播稻育种耐淹亲本的筛选方法
CN104920089B (zh) * 2015-07-03 2017-06-30 徐州佳禾农业科技有限公司 一种直播稻育种耐淹亲本的筛选方法

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