WO2023219154A1 - 水田からのメタン排出量の削減方法、イネにおけるメタン排出量の調節の程度を判定する判定方法、及びイネの包装品 - Google Patents

水田からのメタン排出量の削減方法、イネにおけるメタン排出量の調節の程度を判定する判定方法、及びイネの包装品 Download PDF

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WO2023219154A1
WO2023219154A1 PCT/JP2023/017849 JP2023017849W WO2023219154A1 WO 2023219154 A1 WO2023219154 A1 WO 2023219154A1 JP 2023017849 W JP2023017849 W JP 2023017849W WO 2023219154 A1 WO2023219154 A1 WO 2023219154A1
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rice
deep
methane
rooting
methane emissions
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PCT/JP2023/017849
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English (en)
French (fr)
Japanese (ja)
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岳志 常田
優作 宇賀
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国立研究開発法人農業・食品産業技術総合研究機構
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    • 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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/46—Gramineae or Poaceae, e.g. ryegrass, rice, wheat or maize
    • 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 a method for reducing methane emissions from rice fields, a determination method for determining the degree of adjustment of methane emissions in rice, and rice packaging products.
  • methane gas which has the second largest greenhouse effect after carbon dioxide, is currently attracting attention as an important reduction target in countries such as the United States and China.
  • Rice fields are one of the major sources of methane emissions, accounting for approximately 10% of all methane emissions, and methane reduction is expected to be highly effective in preventing global warming. Therefore, it is desired to develop methane reduction technology that can be easily disseminated to general farmers.
  • Non-Patent Document 1 describes that methane emissions from rice fields can be reduced by extending mid-drying.
  • Non-Patent Document 2 describes a method of reducing methane emissions from rice fields using intermittent irrigation technology (Alternate wetting and drying, AWD).
  • Non-Patent Documents 1 and 2 often impose a burden on producers.
  • the burden includes additional water management work and psychological stress during water shortages.
  • the success or failure of water management is affected by the weather, the drainage characteristics of rice fields, etc., and there is a problem that the effect of reducing methane emissions is unstable.
  • One aspect of the present invention has been made to solve the above-mentioned problems, and aims to reduce methane emissions during rice cultivation in paddy fields.
  • a method for reducing methane emissions from rice fields includes the steps of selecting rice having deep-rooted traits and growing the selected rice. include.
  • a determination method is a method for determining the degree of regulation of methane emission in rice, using whether or not a test rice has deep-rooted traits as an indicator. and determining the degree of amount adjustment.
  • a packaged product of rice having a trait that reduces methane emissions according to one embodiment of the present invention is a package containing a plurality of rice plants that have been determined to have deep rooting traits by the determination method according to one embodiment of the present invention. be.
  • DNA can also be referred to as “polynucleotide,””nucleicacid,” or “nucleic acid molecule,” and is intended to be a polymer of nucleotides.
  • base sequence can also be referred to as a “nucleic acid sequence” or a “nucleotide sequence,” and unless otherwise specified, a deoxyribonucleotide sequence or a ribonucleotide sequence is intended.
  • protein can also be referred to as "polypeptide.”
  • methane emissions refers to the amount of methane gas emitted from paddy fields into the atmosphere when rice is cultivated under flooded conditions. It can also be expressed as the amount of methane gas generated. Furthermore, the “methane emission amount” may also be the methane gas emission amount (flux) per unit time/unit area or the methane gas emission amount per rice yield.
  • rice includes rice varieties such as IR64, Koshihikari, Kinandang, and Patong.
  • a and/or B is a concept that includes both A and B and A or B, and can also be translated as "at least one of A and B.”
  • a method for reducing methane emissions from rice paddies includes the steps of selecting rice having deep-rooted traits and growing the selected rice. This makes it possible to reduce methane emissions from rice fields.
  • the present inventors have found that methane emissions from paddy fields are reduced when rice with deep-rooted traits is cultivated, compared to when rice without deep-rooted traits is cultivated, The present invention was completed based on this new knowledge.
  • the term "deep-rooted trait” refers to the property that roots, which are underground organs of a plant, extend underground at a deep angle with respect to the ground surface.
  • deep angle means that when the ground surface is horizontal and the vertical direction is 90 degrees, the proportion of roots whose extension angle is at least 50 degrees or more is 50% or more of the total number of roots, preferably This means 60% or more, more preferably 70%, even more preferably 80% or more.
  • varieties exhibiting a deep-rooted phenotype are selected from rice varieties.
  • a rice variety may be selected based on a certain rice variety and whether or not the roots grow at a deeper angle than that of the rice variety. Further, in the selection step, a rice variety may be selected based on the root angle compared to a reference rice variety.
  • the method for reducing methane emissions from rice fields is to reduce methane emissions from rice fields compared to the standard methane emissions from rice fields. This will reduce methane emissions from rice fields.
  • the method for reducing methane emissions from rice fields reduces methane emissions from rice fields compared to the case of cultivating rice that does not have deep-rooted traits.
  • the concept of methane emission amount includes the degree of methane emission amount, which indicates whether the methane emission amount of a certain individual rice plant is relatively large or small compared to other rice plants.
  • Methane gas is attracting attention as a reduction target to achieve decarbonization and prevent global warming.
  • Paddy fields where rice is grown, are one of the major sources of methane emissions. Therefore, there is a need for technology that can efficiently and easily reduce methane emissions from rice fields.
  • the method for reducing methane emissions from rice fields by selecting rice plants that have the deep-rooted trait, it is possible to select rice plants that have the trait that reduces methane emissions. Therefore, methane emissions from rice fields can be reduced simply by selecting and cultivating individuals with traits that reduce methane emissions.
  • it is possible to reduce methane emissions from rice fields by simply cultivating the plants selected in the method for reducing methane emissions from rice fields, so it is possible to stably obtain a reduction effect and implement reduction measures. It is easy to verify whether the
  • rice plants having a deep rooting control gene in its genome that encodes a protein having the activity of conferring the deep rooting trait may be selected.
  • the deep-rooted control gene may be a gene involved in determining methane emission (methane emission control gene).
  • a rice variety known in advance to have a deep rooting control gene in its genome may be selected.
  • the deep rooting control gene is the DNA described in any of the following (a) to (c): (a) DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 or 2; (b) DNA consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 or 2, and encoding a protein having the activity of imparting deep rooting traits to rice. ; (c) Consists of an amino acid sequence in which 25 or less amino acids are substituted, deleted, added, or inserted with respect to the amino acid sequence set forth in SEQ ID NO: 1 or 2, and has the activity of imparting deep rooting traits to rice.
  • DNA encoding proteins This is a gene containing
  • Examples of deep-rooted control genes include the DRO1 gene and the qSOR1 gene. Details of the deep rooting control genes will be described later.
  • the rice plant having a deep rooting control gene in its genome may be selected.
  • one example of a method for determining whether or not a rice plant has a deep rooting control gene in its genome is a method for determining the degree of regulation of methane emission in rice, which will be described later.
  • the rice selected in the step of selecting rice is grown.
  • the selected rice is grown by a conventionally known method.
  • the selected rice is grown in a paddy field under irrigation.
  • methane emissions from paddy fields are reduced.
  • methods for reducing methane emissions from paddy fields include information on the reduction in methane emissions during the cultivation of rice with deep-rooted traits compared to the standard methane emissions from paddy fields.
  • a methane emission reduction amount management database including the following for each variety of rice may be obtained, and the rice to be grown may be selected by referring to the methane emission reduction amount management database.
  • Such a database may be constructed in advance and made available to rice producers. Such a database can be suitably used in the emissions trading market regarding methane emissions. By referring to this database and selecting and cultivating rice with deep-rooted traits, rice producers can obtain carbon credits as compensation for reducing methane emissions, and use the obtained carbon credits as emissions credits. Earn money by trading in the trading market.
  • the method for reducing methane emissions from rice fields is to calculate the methane emission reduction of rice grown by referring to the information on the methane emission reduction, and convert the calculated methane emission reduction into carbon credits. Further steps may be included.
  • carbon credits as compensation for reduced methane emissions are granted by a carbon credit certification body, and can be traded in an emissions trading market approved by the certification body.
  • producers of rice endowed with the deep-rooted trait can obtain added value equivalent to the carbon credits by trading the carbon credits obtained by cultivating the rice.
  • a sales entity that sells rice to which deep-rooted traits have been imparted can add the above-mentioned added value to the selling price of the rice and sell it.
  • rice producers can reduce methane emissions during rice cultivation.
  • rice that is registered in the database as having reduced methane emissions is cultivated, it can be determined that methane reduction effects have been achieved, so rewards should be given fairly in accordance with methane emission reductions. I can do it. As a result, it can also contribute to the stability of transactions in the emissions trading market.
  • a method for determining the degree of regulation of methane emissions in rice determines whether methane emissions have decreased or not in rice.
  • the determination method is to determine whether methane emissions have decreased or not by determining whether rice has a deep-rooted trait or not.
  • the determination method includes the step of determining the degree of regulation of methane emissions of the test rice, using as an index whether or not the test rice has a deep-rooted trait. In the determining step, it is determined whether or not the test rice shows a deep-rooted phenotype. In the determination step, the degree of regulation of the methane emission amount of rice may be determined based on a certain rice variety as a reference and whether the roots grow at a deeper angle than that of the rice variety. Further, in the determination step, the degree of adjustment of the methane emission amount of rice may be determined based on the angle of the roots compared to a reference rice variety.
  • the test rice in the determination method may be a rice plant in which it is unclear whether or not it has the deep-rooted trait.
  • the test rice may be a candidate plant for breeding material or a plant obtained through a breeding process.
  • Candidate plants for breeding materials include, for example, parent plants used for hybridization and plants used for molecular breeding using genetic recombination technology.
  • examples of plants obtained through the breeding process include plants resulting from intraspecific hybridization of rice, and their progeny lines.
  • rice may be a plant that is a cross between varieties, such as a hybrid plant between rice belonging to a certain variety and rice belonging to another variety, or a progeny line thereof.
  • the rice may be a plant obtained by crossing varieties known to have deep-rooted traits, or a progeny line thereof.
  • rice may be a plant obtained by crossing a variety known to have deep rooting traits with a variety whose presence is unknown or not, and its progeny lines.
  • rice may be a plant obtained by crossing varieties of which it is unknown whether they have deep-rooted traits, or a progeny line thereof.
  • rice is a plant that is a cross between a variety known to have deep-rooted traits and a variety known not to have deep-rooted traits, and its progeny lines. Good too.
  • rice may be a plant obtained by crossing individuals known to have deep-rooted traits, or a progeny line thereof.
  • a plant may be part or all of a plant.
  • examples of the part of the plant include propagation materials (eg, leaves, branches, seeds, etc.).
  • the test rice has a deep rooting control gene in its genome that encodes a protein with the activity of imparting the deep rooting trait is used as an indicator to determine the regulation of methane emissions in the rice.
  • the degree may also be determined.
  • the genotype of a deep rooting control gene present in the genome of the test rice is determined, thereby determining the rice plant based on its deep rooting ability.
  • the determination method is to use the DNA described in any of the following (a) to (c) in the test rice: (a) DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 or 2; (b) DNA consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 or 2, and encoding a protein that has the function of imparting deep rooting traits to rice. ; (c) Consists of an amino acid sequence in which 25 or less amino acids are substituted, deleted, added or inserted to the amino acid sequence set forth in SEQ ID NO: 1 or 2, and has the function of imparting deep rooting traits to rice.
  • DNA encoding proteins The method may include a step of examining whether or not a deep-rooted control gene containing the deep-rooted control gene or a protein encoded by the deep-rooted control gene is expressed.
  • a DNA sample is prepared from the test rice, (ii) a region containing the deep-rooted regulatory gene is amplified from the DNA sample, and (iii) the molecular weight or base sequence of the amplified DNA fragment is determined. is compared with the molecular weight or base sequence of the deep-rooted control gene.
  • Preparation of DNA samples from test rice can be performed by methods known to those skilled in the art.
  • a preferable preparation method includes, for example, a method of extracting DNA using the CTAB method.
  • the DNA sample used in the determination method is not particularly limited, but usually genomic DNA extracted from test rice is used.
  • the source of genomic DNA is not particularly limited, and it can be extracted from any tissue of rice. Genomic DNA can be extracted from, for example, ears, leaves, roots, stems, seeds, endosperm, bran, embryos, etc., but is not limited thereto.
  • Amplification of a region containing a deep-rooted regulatory gene in a DNA sample can be performed using a primer set that amplifies a region containing DNA described in any one of (a) to (c) above. That is, in the determination method, in the testing step, a primer set that amplifies a region containing the DNA described in any one of (a) to (c) above is used to amplify the region in the DNA of the test rice.
  • the primer set used in PCR is not particularly limited as long as it can amplify the DNA fragment of the target region, and the primer set may be designed so that the length of the amplified fragment is shortened.
  • a primer set is designed such that the length of the primer amplified fragment is preferably 700 base pairs (bp) or less, 200 bp or less, 150 bp or less, 120 bp or less, or 100 bp or less.
  • the primer set includes a first primer that is a forward primer and a second primer that is a reverse primer.
  • the length of these primers may be, for example, 15 bp or more, 16 bp or more, 17 bp or more, 18 bp or more, or 19 bp or more, and may be 50 base bp or less, 40 bp or less, or 30 bp or less.
  • the region to be amplified is a portion corresponding to the genomic DNA region of the deep-rooted control gene. Further, the full length of the genomic DNA may be amplified, or the ORF region encoding the deep rooting control protein (for example, the DNA region set forth in SEQ ID NO: 3 or 4) or a part of the ORF region may be amplified.
  • the region to be amplified may be a region that includes at least a molecular marker for testing the presence or absence of a mutation in a deep-rooted control gene.
  • molecular markers include SNP markers, AFLP (molecular amplified fragment length polymorphism) markers, RFLP markers, microsatellite markers, SCAR markers, and CAPS markers.
  • the region to be amplified when the region to be amplified is part of the DRO1 gene, it may be a region including the 354th base in the base sequence of SEQ ID NO: 3, as an example.
  • the region to be amplified when the region to be amplified is part of the qSOR1 gene, it may be a region containing the 217th base in the base sequence of SEQ ID NO: 4, as an example.
  • Amplification of the region in the DNA of the test rice can be performed by polymerase chain reaction (PCR) using DNA extracted from the rice test subject as a template and primers that amplify the region.
  • PCR polymerase chain reaction
  • PCR can be carried out by those skilled in the art by appropriately selecting reaction conditions and the like.
  • the amplified DNA product can be labeled by using a primer labeled with a 32 P isotope, a fluorescent dye, biotin, or the like.
  • PCR may be either singleplex PCR, which amplifies a DNA fragment with a reaction system containing a single primer set, or multiplex PCR, which amplifies genes with a reaction system containing multiple primer sets.
  • multiplex PCR primer sets labeled with fluorescent substances having different wavelengths (eg, NED, 6-FAM, VIC, PET) may be mixed.
  • PCR reaction conditions can be appropriately set depending on the type of DNA polymerase and PCR device used, the length of the amplified fragment, etc.
  • As the cycle conditions a 3-step PCR method in which one cycle consists of the three steps of denaturation, annealing, and extension, and a two-step PCR method in which one cycle consists of the two steps of denaturation, annealing, and extension are applied. can do.
  • An example of PCR reaction conditions is 90-100°C for 40-60 seconds (e.g., 95°C for 50 seconds), followed by 90-100°C (e.g., 95°C) for 5 seconds, annealing for 10-20 seconds (e.g.
  • PCR reaction conditions may be adjusted in order to stably detect the target region.
  • real-time PCR such as TaqMan (registered trademark)-PCR method or Tm-shift genotyping method (Fukuoka et al., Breed Sci 58:461-464, 2008) may be used. That is, a TaqMan (registered trademark) probe may be used.
  • a high-throughput discrimination method can be provided.
  • Comparison of the molecular weights or base sequences of the amplified DNA fragments can be performed by methods known to those skilled in the art, such as DNA sequencing such as the Sanger method.
  • a DNA fragment can be denatured by applying heat or the like, and then electrophoresed using a polyacrylamide gel containing a denaturing agent such as urea or SDS.
  • SDS-PAGE using SDS as a denaturing agent is an advantageous separation technique in the present invention, and SDS-PAGE can be performed according to the method of Laemmli.
  • the amplified DNA fragment may be analyzed by determining the base sequence using an automatic DNA sequencer or the like.
  • DNA fragments can be detected by staining the gel after electrophoresis using silver staining or the like. For example, it is possible to determine whether a test rice has a deep-rooting trait by amplifying DNA fragments from a rice variety and a test rice plant having the deep-rooting trait using primers and comparing their molecular weights. If the molecular weights match, the test rice is determined to have deep rooting traits.
  • the base sequence of the DNA region of the test rice corresponding to the deep rooting control gene is directly determined and compared with the base sequence of a cultivar with the deep rooting trait. It can be determined whether or not the plant has deep-rooted traits. If the base sequences match, the test plant is determined to have deep rooting traits.
  • matching means that the molecular weights or base sequences of both genes of the alleles match those of a plant having a deep rooting trait, or that the amino acid sequences match. Therefore, if the molecular weight, nucleotide sequence, or amino acid sequence of one of the alleles is different from that of a plant with a deep-rooting trait, but the other is the same as that of a plant with a deep-rooting trait, it is considered to be a "match”. Not included in "Do”.
  • the determination method consists of the base itself (SNP) corresponding to the 354th base of the DNA consisting of the base sequence shown in SEQ ID NO: 3 or the 217th base in the base sequence of SEQ ID NO: 4, or continuous DNA containing the base.
  • SNP base itself
  • Molecular markers may be used to test for the presence or absence of mutations in deep-rooted regulatory genes. Such molecular markers are also included within the scope of the present invention.
  • a packaged product according to one aspect of the present invention is a packaged product of rice having a trait that reduces methane emissions.
  • the packaged product is a package of a plurality of rice plants that have been determined to have deep roots using the determination method according to one aspect of the present invention.
  • the packaged product is a package containing a plurality of rice plants that have been determined to have a deep rooting control gene in its genome that encodes a protein that has the activity of imparting the deep rooting trait.
  • the rice contained in the packaged product may be, for example, seedlings, seeds, or the like.
  • the packaged product may state that the rice included in the packaged product has a trait that reduces methane emissions. Rice producers can reduce methane emissions during rice cultivation simply by obtaining the packaged product and cultivating the rice contained in the packaged product.
  • the deep rooting control gene encodes a protein that has the activity of imparting the deep rooting trait to rice (deep rooting control activity).
  • deep-rooted trait refers to the property that roots, which are underground organs of a plant, extend underground at a deep angle with respect to the ground surface.
  • deep angle means that when the ground surface is horizontal and the vertical direction is 90 degrees, the proportion of roots whose extension angle is at least 50 degrees or more is 50% or more of the total number of roots, preferably This means 60% or more, more preferably 70%, even more preferably 80% or more.
  • the protein has an activity that confers a deep rooting trait, the protein is more deeply rooted than a plant in which that activity is reduced or inhibited, and is more deep rooted than a plant in which that activity is reduced or inhibited. Low methane emissions.
  • plants whose activity is reduced or inhibited are more shallow-rooted than plants whose activity is not reduced or inhibited. They emit more methane than plants without.
  • An example of a deep rooting control gene is a deep rooting control gene involved in regulating deep rooting in rice, and is the DNA described in any of the following (a) to (c): (a) DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 or 2; (b) DNA consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 or 2, and encoding a protein that has the function of imparting deep rooting traits to rice. ; (c) Consists of an amino acid sequence in which 25 or less amino acids are substituted, deleted, added or inserted to the amino acid sequence set forth in SEQ ID NO: 1 or 2, and has the function of imparting deep rooting traits to rice. DNA that codes for proteins.
  • the DNA encoding the protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 is derived from DRO1 (DEEPER) of the rice variety Kinandang Patong. ROOTING 1) This is DNA that contains the base sequence of the open reading frame (ORF) of the gene (SEQ ID NO: 3) and encodes a protein that has the function of imparting the deep rooting trait to rice.
  • DRO1 DEEPER
  • ROOTING 1 This is DNA that contains the base sequence of the open reading frame (ORF) of the gene (SEQ ID NO: 3) and encodes a protein that has the function of imparting the deep rooting trait to rice.
  • the DNA encoding the protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 is based on the ORF base sequence of the qSOR1 (quantitative trait locus for SOIL SURFACE ROOTING 1) gene of rice variety IR64.
  • This DNA contains SEQ ID NO: 4) and encodes a protein that has the function of imparting deep rooting to rice.
  • genes that have the activity of imparting deep rooting traits to rice include the DRO1 gene and the qSOR1 gene. It is described in References 1 and 2 that these genes have the activity of imparting deep rooting traits to rice (Reference 1: International Publication No. WO2011/078308, Reference 2: PNAS , vol. 117, no. 35, 21242-21250). These documents are incorporated herein by reference in their entirety. The present inventors discovered for the first time that the DRO1 gene and the qSOR1 gene, which have the activity of imparting the deep-rooting trait to rice, have the activity of imparting the deep-rooting trait to rice.
  • the sequence identity of the amino acid sequence is preferably 70% or more, 75% or more, 80% or more, or 90% or more, more preferably 95% or more, and 96% or more. % or more, 97% or more, 98% or more, or 99% or more is particularly preferable.
  • a mutant gene derived from rice is included in the category of DNA in (b) above. These mutant genes may be endogenous genes of rice.
  • the number of substituted, deleted, added or inserted amino acids in the amino acid sequence of SEQ ID NO: 1 or 2 is preferably 1 to 25, preferably 1 to 20, 1 The number is preferably 1 to 15, 1 to 10, or 1 to 5, and even more preferably 1 to 4, 1 to 3, or 1 to 2.
  • the deep-rooted control gene refers to a gene into which a mutation has been artificially introduced
  • the above-mentioned "amino acid substitution, deletion, addition, or insertion” is, for example, the Kunkel method (Kunkel et al. (1985): Proc. Natl. Site-specific mutagenesis methods such as Acad. Sci. USA, vol. 82, p488-492, 1985), mutagen treatment using drugs, and mutagenesis by irradiation with radiation ( ⁇ rays, heavy ion beams, etc.) Mutations may be introduced artificially using techniques or the like, or may be derived from similar naturally occurring mutated polypeptides.
  • a deep-rooted control gene can exist in the form of RNA (eg, mRNA) or in the form of DNA (eg, cDNA or genomic DNA). DNA may be double-stranded or single-stranded.
  • the deep-rooted control gene may contain the ORF of the DRO1 gene or the qSOR1 gene, as well as additional sequences such as the base sequence of the untranslated region (UTR).
  • the method for obtaining (isolating) the deep rooting control gene is not particularly limited, but for example, a probe that specifically hybridizes with a part of the base sequence of the deep rooting control gene is prepared, and genomic DNA is isolated. Libraries or cDNA libraries may be screened.
  • a method for obtaining a deep-rooted control gene a method using amplification means such as PCR can be mentioned.
  • primers are prepared from the 5' and 3' sequences (or their complementary sequences) of the cDNA of the deep-rooted regulatory gene, and these primers are used to convert genomic DNA (or cDNA) etc. into templates.
  • genomic DNA or cDNA
  • PCR PCR or the like to amplify the DNA region sandwiched between both primers, a large amount of DNA fragments containing the deep rooting control gene can be obtained.
  • chemically synthesized DNA can be produced using, for example, a commercially available oligonucleotide synthesizer.
  • whether or not the isolated candidate gene for the deep rooting control gene has the desired deep rooting ability can be evaluated by observing whether deep rooting ability is induced by the expression of the candidate gene in the plant from which it is derived. can do.
  • Deep root control genes can be used to elucidate the mechanism that reduces methane emissions in rice. Furthermore, the deep rooting control gene can be used to create transformants by incorporating its sequence into an expression vector and introducing it into rice plants or cells. By cultivating rice into which a deep rooting control gene has been introduced, rice with deep rooting traits can be obtained.
  • the deep rooting control protein is a translation product of the gene described in the deep rooting control gene section above, and has the activity of imparting the deep rooting trait to rice. As mentioned above, the deep rooting regulatory protein does not cause the appearance of the deep rooting trait or inhibit the appearance of the trait.
  • Deep root regulatory proteins may be isolated from natural sources or may be chemically synthesized. More specifically, the proteins include natural purified products, products of chemical synthetic procedures, and prokaryotic or eukaryotic hosts (e.g., bacterial cells, yeast cells, higher plant cells, insect cells, and mammalian cells). This category includes translation products produced by recombinant techniques from animals (including animal cells).
  • prokaryotic or eukaryotic hosts e.g., bacterial cells, yeast cells, higher plant cells, insect cells, and mammalian cells. This category includes translation products produced by recombinant techniques from animals (including animal cells).
  • the deep rooting control protein can be obtained, for example, by inserting the deep rooting control gene into an appropriate expression vector, introducing the vector into appropriate cells, culturing the transformed cells, expressing it, and purifying it.
  • Recombinant proteins expressed in host cells can be purified and recovered from the host cells or their culture supernatants by methods known to those skilled in the art. It is also possible to produce a transformed plant into which a deep rooting control gene has been introduced using the method described below, and to prepare a deep rooting control protein from the plant.
  • the deep rooting regulatory protein is a protein described in any one of the following (a') to (c'): (a') a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 or 2; (b') A protein consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 or 2, and having the function of imparting a deep rooting trait to rice; (c') Consists of an amino acid sequence in which 25 or less amino acids are substituted, deleted, added or inserted to the amino acid sequence set forth in SEQ ID NO: 1 or 2, and has the function of imparting deep rooting traits to rice. protein with.
  • the protein (a') above is a protein encoded by the DRO1 gene or the qSOR1 gene, and is a protein that has the activity of imparting deep rooting traits to rice.
  • the sequence identity of the amino acid sequence is preferably 70% or more, 75% or more, 80% or more, or 90% or more, more preferably 95% or more, and 96% or more. % or more, 97% or more, 98% or more, or 99% or more is particularly preferable.
  • a mutant protein derived from rice is included in the category of protein (b') above. These mutant proteins are proteins encoded by endogenous genes of rice.
  • the number of substituted, deleted, added or inserted amino acids in the amino acid sequence of SEQ ID NO: 1 or 2 is preferably 1 to 25, preferably 1 to 20, 1 The number is preferably 1 to 15, 1 to 10, or 1 to 5, and even more preferably 1 to 4, 1 to 3, or 1 to 2.
  • a deep-rooted regulatory protein is a polypeptide formed by peptide bonding of amino acids, but it may also contain a structure other than a polypeptide. Structures other than polypeptides herein include, but are not limited to, sugar chains and isoprenoid groups.
  • An expression vector into which a deep rooting control gene has been integrated, a cell containing the expression vector or the deep rooting control gene, and a transformant into which the expression vector or the deep rooting control gene is introduced to be able to express the deep rooting control gene are It can be used to produce rice that has this in its genome.
  • the expression vector is used to produce deep rooting regulatory proteins. That is, the expression vector imparts deep-rooted traits to cells or individual organisms.
  • the type of vector used to construct the expression vector is not particularly limited, and one that can be expressed in host cells may be appropriately selected. That is, an appropriate promoter sequence may be selected depending on the type of host cell, and the promoter sequence and deep rooting control gene may be incorporated into, for example, a plasmid, a phagemid, or a cosmid, and used as an expression vector.
  • Examples of host cells into which the expression vector is introduced include bacterial cells, yeast cells, fungal cells other than yeast cells, and higher eukaryotic cells.
  • Examples of bacterial cells include E. coli cells.
  • Higher eukaryotic cells include, for example, plant cells and animal cells.
  • Plant cells include, for example, dicot cells and monocot cells.
  • Examples of dicotyledonous plant cells include suspension-cultured cells of plants of the Solanaceae family (eg, tobacco BY-2 strain and tomato Sly-1 strain).
  • Examples of monocotyledonous plant cells include the Oc strain, which is a suspension cultured cell of rice.
  • Examples of animal cells include insect cells, amphibian cells, reptile cells, avian cells, fish cells, and mammalian cells.
  • elements necessary for transcription are functionally linked to the deep-rooted control gene.
  • an enhancer, selection marker, splicing signal, polyA addition signal, 5'-UTR sequence, etc. may be linked as necessary.
  • a promoter is a DNA sequence that exhibits transcriptional activity in host cells, and can be appropriately selected depending on the type of host.
  • the deep-rooted control gene may be operably linked to an appropriate terminator (eg, NOS terminator and cauliflower mosaic virus 35S terminator), if necessary.
  • an appropriate type of terminator may be appropriately selected depending on the type of host cell, and any sequence that can terminate the transcription of the gene transcribed by the above-mentioned promoter may be used.
  • Enhancers are used to increase the expression efficiency of a target gene, and include, for example, the omega sequence of tobacco mosaic virus.
  • the expression vector may further contain a selection marker.
  • Selectable markers can include, for example, drug resistance genes such as ampicillin, kanamycin, tetracycline, chloramphenicol, neomycin, hygromycin or spectinomycin.
  • the deep-rooted control gene may be linked to a suitable tag sequence for protein purification or a suitable spacer sequence, as necessary.
  • the cells capable of expressing the deep rooting control gene may be cells into which an expression vector incorporating the deep rooting control gene has been introduced, and can be used as a production system for producing or expressing the deep rooting control protein.
  • plant-derived cells When using plant cells as hosts, plant-derived cells may be used and cultured as callus.
  • Various methods known to those skilled in the art can be used to introduce the expression vector into host cells. For example, to introduce expression vectors into E. coli cells, it is also possible to use an introduction method using calcium ions, and to introduce expression vectors into plant cells, polyethylene glycol method and electroporation method can be used. ) etc. can be used.
  • transformant includes not only cells, tissues, and organs into which the expression vector or deep-rooted control gene is introduced so that it can be expressed, but also an individual organism.
  • a transformant may be rice.
  • the transformant may be, for example, a microorganism such as E. coli, an animal, or the like.
  • Rice having deep-rooted traits is a plant obtained by the production method described below. Rice plants with deep rooting traits have deep rooting control genes.
  • rice with deep-rooted traits is produced from plants obtained by crossing rice with deep-rooted traits with other rice plants and their progeny lines. This can be obtained by identifying rice plants with deep-rooted traits using a method for determining the degree of regulation of methane emissions]. Note that rice plants having deep rooting traits that have been genetically modified to have deep rooting control genes are also included in the scope of the present invention.
  • a method for producing rice having a deep-rooting trait includes the steps of executing a determination method for determining the degree of regulation of methane emission in rice according to one embodiment of the present invention, and selecting rice having a deep-rooting trait. .
  • the rice production method further includes a step of intraspecific crossing of rice having deep rooting traits with another rice, and in the selection step, the rice obtained by the intraspecific crossing or its progeny.
  • Rice having deep-rooted traits may be selected from the rice lines.
  • the rice plant with the deep-rooting trait used as the parent plant may be the rice plant with the deep-rooting trait according to one embodiment of the present invention.
  • the rice with the deep-rooting trait used in the crossing step is the rice with the deep-rooting trait selected by the determination method for determining the degree of regulation of methane emission in rice according to one embodiment of the present invention. It's okay. That is, in the rice production method, before the crossbreeding step, rice having deep-rooted traits is extracted from the test rice using the determination method for determining the degree of regulation of methane emission in rice according to one aspect of the present invention. It may further include a selection step of selecting.
  • a method for producing rice having deep-rooted traits includes, in rice, using the DNA according to any one of the following (a) to (c) or an expression vector containing the DNA: (a) DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 or 2; (b) DNA consisting of an amino acid sequence having 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 or 2, and encoding a protein that has the function of imparting deep rooting traits to rice. ; (c) Consists of an amino acid sequence in which 25 or less amino acids are substituted, deleted, added or inserted to the amino acid sequence set forth in SEQ ID NO: 1 or 2, and has the function of imparting deep rooting traits to rice. DNA encoding proteins; The method includes the step of introducing into rice cells.
  • a preferred embodiment of the rice production method includes a production method for producing a transformed rice plant having a deep-rooted trait by the following steps (I) and (II). Received: (I) A step of introducing a deep rooting regulatory gene or an expression vector containing the gene into plant cells; (II) A step of regenerating a plant from the plant cells of step (I).
  • the step of introducing the deep rooting control gene or the expression vector containing the gene into plant cells in (I) above can be carried out by the method described in the section [Expression vectors, cells, and transformants] above. .
  • the regeneration of a plant from plant cells in (II) above can be carried out by methods known to those skilled in the art depending on the type of plant. Examples include, but are not limited to, the method described in Fujimura et al. (Fujimura et al. Tissue Culture Lett. 2, 1995, 74.).
  • Whether or not a regenerated plant has a deep-rooted trait can be determined by a determination method for determining the degree of regulation of methane emissions in rice according to one embodiment of the present invention.
  • the rice production method if a transformed plant in which the deep rooting control gene of the present invention is introduced into the chromosome is obtained, it is possible to obtain offspring from the plant by sexual or asexual reproduction.
  • cells and propagation materials e.g., seeds, fruits, cuttings, stems, roots, stocks, callus, protoplasts, etc.
  • mass production is also possible.
  • these plant cells, plants containing the cells, progeny and clones of the plants, and propagation materials of the plants, progeny and clones can be used to impart deep rooting traits to plants. It is possible.
  • Figure 1 shows differences in root elongation angles depending on the DRO1 gene and/or qSOR1 gene.
  • the root elongation angle differed depending on the DRO1 gene and/or the qSOR1 gene, as shown in FIG. 1. That is, a line in which both of these genes were functional was a deep root type, and a line homozygous for the function-defective DRO1 and function-defective qSOR1 genes was an ultra-shallow root type. Lines in which one of the strains is functional are intermediate between them, and among them, DRO1(+)/qSOR1(-) is a deeper-rooted type than DRO1(-)/qSOR1(+).
  • DRO1( ⁇ )/qSOR1(+) is an allele originally possessed by IR64, and was regarded as a control in this example. As these four lines, the four lines described in FIG. 3 of Reference Document 2 were used.
  • Methane emissions were measured using a closed chamber method (Minamikawa et al. 2015. ISBN:978-4-931508-16-3 (online)). Using an acrylic chamber with a bottom area of 30 cm x 60 cm, which corresponds to the area of 4 plants, the amount of methane accumulated in the chamber was analyzed using a gas chromatograph equipped with an FID detector. The amount of discharge (flux) was measured.
  • Figure 2 shows the temporal changes in methane emissions of four rice lines with different DRO1/qSOR1 genes, and the numbers at the bottom of the figure are for deep-rooted lines (DRO1 (+)/qSOR1(+)) represents the change (%).
  • Figure 3 shows the difference between systems in cumulative methane emissions over the entire measurement period, and the numbers on the figure represent changes in emissions from each system when compared to IR64 (DRO1(-)/qSOR1(+)). (%) A deep-rooted line with functional alleles of DRO1 and qSOR1 grown from paddy rice cultivar IR64 had lower methane emissions in the latter half of growth (Figure 2), and emissions throughout the growing period were 14% lower than in IR64. ( Figure 3). Conversely, methane emissions increased in shallow-rooted lines (DRO1(-), qSOR1(-)) than IR64 ( Figures 2 and 3).
  • Figure 4 shows (left) polished brown rice yield (15% moisture) of four lines with different DRO1/qSOR1 genes, and the numbers on the figure represent the change (%) of each line when compared with IR64.
  • Figure 5 shows the methane emissions per yield, and the numbers on the figure represent the change (%) of each line when compared to IR64.
  • the yield of the deep-rooted line (DRO1(+)/qSOR1(+)) was the same as that of IR64 (+0.3%, Figure 4), so methane emissions were suppressed (-15%) while maintaining the yield. , Figure 5).
  • FIG. 6 shows changes in the concentration of divalent iron (Fe(II)) in soil water for four lines with different DRO1/qSOR1 genes.
  • the divalent iron concentration was suppressed to a lower level throughout the growing period compared to IR64.
  • the soil becomes relatively oxidative, suppressing the production of methane by microorganisms, and promoting methane oxidation around the oxidative rice roots, resulting in a reduction in methane emissions. It was thought that this led to (Figure 6).
  • the present invention can be used not only in the agricultural field, but also in the environmental field through the maintenance and development of agriculture.

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