Method for reducing methane emissions from paddy fields, determination method for determining degree of regulation of methane emissions in rice plant, and rice packaging product

Abstract

To reduce methane emissions during cultivation of rice plants.SOLUTION: This method for reducing methane emissions from paddy fields comprises a step of selecting a rice plant having the trait of deep rooting, and a step of growing the selected rice plant.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for reducing methane emissions from paddy fields, a method for determining the degree of control of methane emissions in rice plants, and packaged rice products. [Background technology]

[0002] Since the Paris Agreement came into effect in 2016, efforts toward decarbonization and global warming prevention have been accelerating worldwide. Within this trend toward decarbonization and global warming prevention, 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. Paddy fields are one of the major sources of methane emissions, accounting for approximately 10% of total methane emissions, and methane reduction is expected to be highly effective in preventing global warming. Therefore, there is a need for the development of methane reduction technologies that can be easily adopted by ordinary farmers.

[0003] One known method for reducing methane emissions from rice paddies is to make the paddy soil aerobic through water management. Non-Patent Document 1 describes reducing methane emissions from rice paddies by extending mid-season drainage. Non-Patent Document 2 describes a method for reducing methane emissions from rice paddies using intermittent irrigation technology (alternate wetting and drying, AWD). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Itoh et al., Mitigation of methane emissions from paddy fields by prolonging midseason drainage. Agriculture, Ecosystems & Environment, 141, 359-372, 2011 [Non-patent document 2] Tirol-Padre, A. et al., Site-specific feasibility of alternate wetting and drying as a greenhouse gas mitigation option irrigated rice fields in Southeast Asia:a synthesis. Soil Science and Plant Nutrition,64,2-13,2018 Summary of the Invention [Problem to be solved by the invention]

[0005] However, the cultivation techniques described in Non-Patent Documents 1 and 2 often impose a burden on producers. These burdens include additional water management work and psychological stress during water shortages. Furthermore, the success of water management is affected by factors such as weather and the drainage of paddy fields, making the effectiveness of methane emission reductions unstable. Furthermore, when establishing a subsidy system for producers who contribute to methane reduction, it is difficult to verify whether the producers actually carried out the management, making it difficult to fairly award rewards based on methane emission reductions. These issues hinder the widespread adoption of efforts to reduce methane emissions from paddy fields.

[0006] One aspect of the present invention has been made to solve the above-mentioned problems, and aims to achieve a reduction in methane emissions during rice cultivation in paddy fields. [Means for solving the problem]

[0007] In order to solve the above problem, one embodiment of the present invention provides a method for reducing methane emissions from paddy fields, which includes the steps of selecting rice plants having deep-rooted traits and growing the selected rice plants.

[0008] A determination method according to one embodiment of the present invention is a method for determining the degree of regulation of methane emission in rice, and includes a step of determining the degree of regulation of methane emission in the test rice using as an indicator whether or not the test rice has a deep-rooted trait.

[0009] A packaged product of rice having a trait that reduces methane emissions according to one embodiment of the present invention is a package of multiple rice plants that have been determined to have the trait of deep rooting using a determination method according to one embodiment of the present invention. [Effects of the Invention]

[0010] According to one aspect of the present invention, it is possible to reduce methane emissions from rice paddies. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 shows the difference in the state of rice roots caused by the DRO1 gene and the qSOR1 gene. [Figure 2] 1 is a graph showing methane emissions from each rice plant in the examples. [Figure 3] 1 is a graph showing the cumulative methane emissions for each rice plant in the examples. [Figure 4] 1 is a graph showing the yield of polished brown rice for each rice plant in the examples. [Figure 5] 1 is a graph showing methane emission per yield for each rice plant in the Examples. [Figure 6] 1 is a graph showing the divalent iron concentration in soil water for each rice plant in the Examples. DETAILED DESCRIPTION OF THE INVENTION

[0012] The following describes an embodiment of the present invention, but the present invention is not limited to this.

[0013] [Definitions of terms, etc.] As used herein, "DNA" can be alternatively referred to as "polynucleotide," "nucleic acid," or "nucleic acid molecule," and refers to a polymer of nucleotides. Furthermore, "base sequence" can be alternatively referred to as "nucleic acid sequence" or "nucleotide sequence," and refers to a sequence of deoxyribonucleotides or a sequence of ribonucleotides, unless otherwise specified. As used herein, "protein" can be alternatively referred to as "polypeptide."

[0014] In this specification, "methane emission" refers to the amount of methane gas emitted into the atmosphere from rice paddies when the rice is grown under flooded conditions. It can also be expressed as the amount of methane gas generated during rice cultivation under flooded conditions. Furthermore, "methane emission" can also refer to the amount of methane gas emitted (flux) per unit time and unit area, or the amount of methane gas emitted per rice yield.

[0015] As used herein, "rice" includes rice varieties such as IR64, Koshihikari, and Kinandang Patong.

[0016] In this specification, "A and / or B" is a concept that includes both A and B and A or B, and can also be expressed as "at least one of A and B."

[0017] [Methods for reducing methane emissions from rice paddies] A method for reducing methane emissions from paddy fields according to one embodiment of the present invention includes the steps of selecting rice plants with deep-rooted traits and growing the selected rice plants. This method enables the reduction of methane emissions from paddy fields. The inventors discovered that cultivating rice plants with deep-rooted traits reduces methane emissions from paddy fields compared to cultivating rice plants without deep-rooted traits, and completed the present invention based on this novel finding. Here, "deep-rooted trait" refers to the property of roots, which are underground organs of a plant, growing underground at a deep angle relative to the soil surface. Furthermore, "deep angle" refers to the proportion of roots with an angle of at least 50 degrees relative to the soil surface of 50% or more of the total number of roots, preferably 60% or more, more preferably 70% or more, and even more preferably 80% or more.

[0018] In the selection step, a variety exhibiting a deep-rooted phenotype is selected from among rice varieties. In the selection step, a rice variety may be selected based on whether the roots of the rice variety grow at a deeper angle than a reference rice variety. In addition, in the selection step, the rice variety may be selected based on the root angle compared to the reference rice variety.

[0019] The method for reducing methane emissions from paddy fields reduces methane emissions from paddy fields compared to the amount of methane emissions from a reference paddy field. This achieves a reduction in methane emissions from paddy fields. The method for reducing methane emissions from paddy fields reduces methane emissions from paddy fields compared to cultivating rice that does not have the deep-rooted trait. Here, the concept of methane emission includes the degree of methane emission, which indicates whether the methane emission of a certain rice plant is relatively high or low compared to other rice plants.

[0020] Methane gas is attracting attention as a target for reduction in order to achieve decarbonization and prevent global warming. Paddy fields, where rice is cultivated, are one of the major sources of methane emissions. Therefore, technologies that can efficiently and easily reduce methane emissions from paddy fields are desired. According to a method for reducing methane emissions from paddy fields, by selecting rice plants with deep root traits, it is possible to select plants with traits that reduce methane emissions. Therefore, methane emissions from paddy fields can be reduced simply by selecting and cultivating plants with traits that reduce methane emissions. Furthermore, since methane emissions from paddy fields can be reduced simply by cultivating the plants selected using this method, the reduction effect can be obtained consistently, and it is easy to verify whether the reduction measures have been implemented.

[0021] In the selection step, rice plants having in their genome a deep-rootedness control gene encoding a protein with activity that confers a deep-rooted trait may be selected. By selecting and growing rice plants having the deep-rootedness control gene in their genome, methane emissions from paddy fields can be reduced. In other words, the deep-rootedness control gene may be a gene involved in determining methane emissions (methane emission control gene). In the rice selection step, for example, rice varieties known in advance to have the deep-rootedness control gene in their genome may be selected.

[0022] The deep rooting control gene is a DNA selected from any one 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) a DNA encoding a protein having an amino acid sequence that has 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 or 2 and has the activity of conferring deep-rooted traits to rice; (c) DNA encoding a protein having an amino acid sequence in which 25 or fewer amino acids have been substituted, deleted, added, or inserted relative to the amino acid sequence set forth in SEQ ID NO: 1 or 2, and having the activity of imparting deep-rooted traits to rice; It is a gene containing

[0023] Examples of deep rooting control genes include the DRO1 gene and the qSOR1 gene. The deep rooting control genes will be described in detail later.

[0024] In the step of selecting rice plants, it is possible to determine whether rice plants, whose genomes are unknown, have the deep-rootedness control gene in their genomes, and select rice plants that have the deep-rootedness control gene in their genomes based on the determination results. In the step of selecting rice plants, an example of a method for determining whether rice plants have the deep-rootedness control gene in their genomes is the method for determining the degree of regulation of methane emission in rice, which will be described later.

[0025] In the step of growing rice, the rice selected in the step of selecting rice is grown. In the step of growing rice, as an example, the selected rice is grown by a conventionally known method. In the step of growing rice, the selected rice is grown under irrigation in a paddy field. In the step of growing rice, by cultivating rice with a deep-rooted trait, methane emissions from the paddy field are reduced.

[0026] In addition, in the method for reducing methane emissions from rice paddies, in the step of selecting rice, a methane emission reduction management database may be obtained that contains information on the amount of methane emission reduction during cultivation of rice with deep-rooted traits compared to the amount of methane emission from a reference rice paddy, for each rice variety, and the rice to be grown may be selected by referring to the methane emission reduction management database.

[0027] Such a database may be constructed in advance and made available to rice producers. Such a database may be suitably used in an emissions trading market for methane emissions. By referring to the database and selecting and cultivating rice plants with deep-rooted traits, rice producers can obtain carbon credits in exchange for reduced methane emissions and trade the carbon credits in the emissions trading market, thereby earning profits. In other words, the method for reducing methane emissions from paddy fields may further include the steps of calculating the amount of methane emission reduction from the rice to be grown by referring to the information on the amount of methane emission reduction, and converting the calculated amount of methane emission reduction into carbon credits.

[0028] Carbon credits, which are compensation for methane emission reductions, are granted by a carbon credit certification organization and can be traded on an emissions trading market approved by the certification organization. This allows producers of rice with deep-rooted traits to trade the carbon credits they earn by cultivating the rice, thereby gaining added value equivalent to the carbon credits. Furthermore, sellers of rice with deep-rooted traits can add the added value to the selling price of the rice.

[0029] Rice producers can reduce methane emissions from rice cultivation by selecting rice varieties with the aim of obtaining these credits. Furthermore, if rice registered in the database as having reduced methane emissions is cultivated, it can be determined that methane reduction effects have been achieved, making it possible to fairly award rewards according to methane emission reductions. As a result, this can also contribute to the stability of transactions in the emissions trading market.

[0030] [Method for determining the degree of regulation of methane emissions in rice] A method for determining the degree of regulation of methane emission in rice according to one embodiment of the present invention (determination method) determines whether methane emission is reduced or not in rice by determining whether the rice has a deep-rooted trait or not.

[0031] A method for determining methane emission regulation according to one embodiment of the present invention includes a step of determining the degree of regulation of methane emission in a test rice plant by using as an index whether the test rice plant has a deep-rooted trait. In the determining step, it is determined whether the test rice plant exhibits a deep-rooted phenotype. In the determining step, the degree of regulation of methane emission in the rice plant may be determined based on whether the roots of a certain rice variety are grown at a deeper angle than those of the reference rice variety. In addition, in the determining step, the degree of regulation of methane emission in the rice plant may be determined based on the root angle compared to that of the reference rice variety.

[0032] The test rice plant in the determination method may be a rice plant whose deep-rooted trait is unknown. The test rice plant may be a candidate plant for breeding material or a plant obtained through a breeding process. Examples of candidate plants for breeding material include parent plants used in crossbreeding and plants used in molecular breeding using genetic engineering techniques. Examples of plants obtained through a breeding process include plants obtained by intraspecific hybridization of rice and their progeny lines. The rice may also be an intervarietal hybrid, such as a hybrid between a rice variety and a rice variety, and its progeny lines. Furthermore, the rice may also be a plant obtained by hybridization between varieties known to have deep-rooted traits, and its progeny lines.

[0033] Furthermore, the rice may be a plant obtained by crossing a variety known to have a deep-rooted trait with a variety whose deep-rooted trait is unknown, and its progeny line. Furthermore, the rice may be a plant obtained by crossing varieties whose deep-rooted trait is unknown, and its progeny line. Furthermore, the rice may be a plant obtained by crossing a variety known to have a deep-rooted trait with a variety whose deep-rooted trait is unknown, and its progeny line. Furthermore, the rice may be a plant obtained by crossing individuals known to have a deep-rooted trait with each other, and its progeny line.

[0034] In this specification, the term "plant" may refer to a part or the whole of a plant body. Examples of a part of a plant body include propagation materials (e.g., leaves, branches, seeds, etc.).

[0035] In the determination step, the degree of regulation of methane emission of the test rice may be determined based on whether the test rice has a deep-rootedness controlling gene in its genome that encodes a protein having activity that confers the trait of deep rootedness. In the determination step, for example, the genotype of the deep-rootedness controlling gene present in the genome of the test rice is determined to determine the rice based on its deep rootedness.

[0036] The determination method comprises the step of detecting a DNA selected from any one of the following (a) to (c) in a test rice plant: (a) DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 or 2; (b) a DNA encoding a protein having an amino acid sequence that has 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 or 2 and that has the function of conferring deep-rooted traits to rice; (c) DNA encoding a protein having an amino acid sequence in which 25 or fewer amino acids have been substituted, deleted, added, or inserted relative to the amino acid sequence set forth in SEQ ID NO: 1 or 2, and having the function of imparting deep-rooted traits to rice; The method may include a step of examining whether or not a deep-rootedness regulatory gene comprising the above-mentioned gene or a protein encoded by the deep-rootedness regulatory gene is expressed.

[0037] In the testing process, for example, (i) a DNA sample is prepared from the test rice, (ii) a region containing the deep-rootedness control gene is amplified from the DNA sample, and (iii) the molecular weight or base sequence of the amplified DNA fragment is compared with the molecular weight or base sequence of the deep-rootedness control gene.

[0038] Preparation of a DNA sample from test rice can be performed by methods known to those skilled in the art. A preferred preparation method is, for example, a method of extracting DNA using the CTAB method. The DNA sample used in the determination method is not particularly limited, but genomic DNA extracted from the test rice is usually 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, panicles, leaves, roots, stems, seeds, endosperm, bran, embryos, etc., but is not limited to these.

[0039] The region containing the deep-rootedness control gene in the DNA sample can be amplified using a primer set that amplifies a region containing the DNA described in any one of (a) to (c) above. That is, in the testing step of the determination method, the region in the DNA of the test rice plant is amplified using a primer set that amplifies a region containing the DNA described in any one of (a) to (c) above.

[0040] The primer set used in PCR is not particularly limited as long as it can amplify a DNA fragment of the target region, and the primer set may be designed so that the length of the amplified fragment is short. For example, the primer set is designed so 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, or may be 50 bp or less, 40 bp or less, or 30 bp or less.

[0041] The region to be amplified corresponds to the genomic DNA region of the deep-rootedness control gene. The entire genomic DNA may be amplified, or the ORF region encoding the deep-rootedness control protein (e.g., the DNA region set forth in SEQ ID NO: 3 or 4) or a portion of the ORF region may be amplified.

[0042] The region to be amplified may contain at least a molecular marker for testing for the presence or absence of a mutation in the deep-rooting control gene, such as a SNP marker, an AFLP (amplified fragment length polymorphism) marker, an RFLP marker, a microsatellite marker, a SCAR marker, or a CAPS marker.

[0043] When the region to be amplified is a part of the DRO1 gene, it may be, for example, a region including the 354th base in the base sequence of SEQ ID NO: 3. When the region to be amplified is a part of the qSOR1 gene, it may be, for example, a region including the 217th base in the base sequence of SEQ ID NO: 4.

[0044] The region in the DNA of the test rice plant can be amplified by polymerase chain reaction (PCR) using DNA extracted from the test rice plant as a template and primers that amplify the region.

[0045] Those skilled in the art can carry out PCR by appropriately selecting reaction conditions, etc. 32 The amplified DNA product can be labeled by using a primer labeled with a P isotope, a fluorescent dye, biotin, or the like.

[0046] PCR may be either singleplex PCR, which amplifies DNA fragments in a reaction system containing a single primer set, or multiplex PCR, which amplifies genes in a reaction system containing multiple primer sets. In the case of multiplex PCR, primer sets labeled with fluorescent substances with different wavelengths (e.g., NED, 6-FAM, VIC, PET) may be mixed.

[0047] PCR reaction conditions can be appropriately set depending on the type of DNA polymerase and PCR instrument used, the length of the amplified fragment, and other factors. Cycling conditions include a three-step PCR method, in which one cycle consists of three steps: denaturation, annealing, and extension; and a two-step PCR method, in which one cycle consists of two steps: denaturation, annealing, and extension. An example of PCR reaction conditions is 90-100°C for 40-60 seconds (e.g., 95°C for 50 seconds), followed by 30-60 cycles (e.g., 40 cycles) of 90-100°C (e.g., 95°C) for 5 seconds, annealing for 10-20 seconds (e.g., 15 seconds), and 65-80°C for 10-30 seconds (e.g., 72°C for 20 seconds). The annealing temperature can be gradually decreased from an initial annealing temperature of 60-70°C (e.g., 66°C) to a final annealing temperature of 50-60°C (e.g., 56°C) every specified cycle. Depending on the state of the template DNA, PCR reaction conditions may be adjusted to stably detect the target region.

[0048] As PCR, real-time PCR such as TaqMan®-PCR or Tm-shift genotyping (Fukuoka et al., Breed Sci 58:461-464, 2008) may be used. That is, TaqMan® probes may be used. The use of real-time PCR can provide a high-throughput discrimination method.

[0049] Comparison of the molecular weight or base sequence of the amplified DNA fragments can be performed by methods known to those skilled in the art, such as DNA sequencing by the Sanger method. For example, the DNA fragments can be denatured by heating or other methods, followed by electrophoresis using a polyacrylamide gel containing a denaturant such as urea or SDS. SDS-PAGE, which uses SDS as a denaturant, is an advantageous separation method in the present invention, and can be performed according to the Laemmli method. Alternatively, the amplified DNA fragments can be analyzed by determining their base sequences using an automated DNA sequencer or the like.

[0050] When labeled DNA is used, the mobility of DNA fragments can be detected and analyzed after electrophoresis using autoradiography with X-ray film or a scanner that detects fluorescence. When labeled DNA is not used, DNA fragments can be detected by staining the gel after electrophoresis using silver staining or other methods. For example, DNA fragments can be amplified from a deep-rooted variety and a test rice plant using primers, and the molecular weights can be compared to determine whether the test rice plant has the deep-rooted trait. If the molecular weights match, the test rice plant is determined to have the deep-rooted trait.

[0051] In addition, when comparing the base sequences of DNA fragments, the base sequence of the DNA region of the test rice corresponding to the deep-rooting control gene can be directly determined and compared with the base sequence of a variety that has the deep-rooting trait, thereby determining whether the test rice has the deep-rooting trait. If the base sequences match, the test plant is determined to have the deep-rooting trait.

[0052] Here, "matching" means that the molecular weight or nucleotide sequence of both genes of an allele matches that of a plant with a deep-rooted trait, or that the amino acid sequence matches that of the plant with a deep-rooted trait. Therefore, if the molecular weight, nucleotide sequence, or amino acid sequence of one gene of an allele is different from that of a plant with a deep-rooted trait, but the other gene is the same as that of a plant with a deep-rooted trait, this does not fall under the category of "matching."

[0053] The determination method may involve testing for the presence or absence of a mutation in the deep-rootedness control gene using a molecular marker consisting of a single nucleotide polymorphism (SNP) corresponding to the 354th base of the DNA consisting of the base sequence shown in SEQ ID NO: 3 or the 217th base of the base sequence shown in SEQ ID NO: 4, or a contiguous DNA sequence containing such a base. Such molecular markers are also included in the scope of the present invention.

[0054] [Packaged goods] A packaged product according to one embodiment of the present invention is a packaged product of rice plants having a trait that reduces methane emissions. The packaged product is a package of multiple rice plants determined to have deep rooting characteristics by the determination method according to one embodiment of the present invention. As an example, the packaged product is a package of multiple rice plants determined to have in their genome a deep rooting control gene that encodes a protein with activity that confers the trait of deep rooting characteristics. The rice plants contained in the packaged product may be, for example, seedlings, seeds, etc. The packaged product may state that the rice plants contained in the packaged product have a trait that reduces methane emissions. Rice producers can achieve reduced methane emissions in rice cultivation simply by obtaining the packaged product and cultivating the rice plants contained in the packaged product.

[0055] [Deep root control gene] The deep-rooting control gene encodes a protein that has the activity to confer the trait of deep rooting to rice (deep-rooting control activity). Here, "deep-rooting trait" refers to the property of the roots, which are underground organs of the plant body, to extend underground at a deep angle relative to the soil surface. Furthermore, "deep angle" means that the proportion of roots with an angle of at least 50 degrees relative to the soil surface is 50% or more, preferably 60% or more, more preferably 70%, and even more preferably 80% or more of the total number of roots.

[0056] When the protein has activity that confers the trait of deep rooting, the plant will have deeper roots than plants in which the activity is reduced or inhibited, and will emit less methane than plants in which the activity is reduced or inhibited.Furthermore, when the activity of the protein is reduced or inhibited, the plant will have shallower roots than plants in which the activity is not reduced or inhibited, and will emit more methane than plants in which the activity is not reduced or inhibited.

[0057] An example of a deep-rooting regulatory gene is a deep-rooting regulatory gene involved in regulating deep-rooting in rice, and is a DNA described in any one of (a) to (c) below: (a) DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 or 2; (b) a DNA encoding a protein having an amino acid sequence that has 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 or 2 and that has the function of conferring deep-rooted traits to rice; (c) DNA consisting of an amino acid sequence in which 25 or fewer amino acids have been substituted, deleted, added, or inserted relative to the amino acid sequence set forth in SEQ ID NO: 1 or 2, and encoding a protein that has the function of conferring deep-rooted traits to rice.

[0058] Among the DNAs in (a) above, the DNA encoding the protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 contains the base sequence (SEQ ID NO: 3) of the open reading frame (ORF) of the DRO1 (DEEPER ROOTING 1) gene of the rice variety Kinandang Patong, and encodes a protein that has the function of conferring deep-rooted traits to rice.

[0059] Among the DNAs in (a) above, the DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 contains the base sequence (SEQ ID NO: 4) of the ORF of the qSOR1 (quantitative trait locus for SOIL SURFACE ROOTING 1) gene of the rice variety IR64, and encodes a protein that has the function of conferring deep rooting to rice.

[0060] Examples of genes that have the activity of conferring deep-rooted traits to rice include the DRO1 gene and the qSOR1 gene. It has been described in References 1 and 2 that these genes have the activity of conferring deep-rooted 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 were the first to discover that the DRO1 gene and the qSOR1 gene, which have the activity of conferring deep-rooted traits to rice, have the activity of conferring deep-rooted traits to rice.

[0061] With respect to the DNA of (b) above, 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 particularly preferably 96% or more, 97% or more, 98% or more, or 99% or more. For example, mutant genes derived from rice are included in the category of DNA of (b) above. These mutant genes may be endogenous genes of rice.

[0062] With respect to the polynucleotide (c) above, 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, more preferably 1 to 20, 1 to 15, 1 to 10, or 1 to 5, and even more preferably 1 to 4, 1 to 3, or 1 to 2.

[0063] In addition, when 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" may be an artificial mutation introduced using, for example, a site-specific mutagenesis method such as the Kunkel method (Kunkel et al. (1985): Proc. Natl. Acad. Sci. USA, vol. 82, p488-492, 1985), mutagen treatment using a drug, or a mutagenesis method using radiation (gamma rays, heavy ion beams, etc.), or may be derived from a similar mutant polypeptide that exists in nature.

[0064] The deep rooting control gene may exist in the form of RNA (e.g., mRNA) or DNA (e.g., cDNA or genomic DNA). The DNA may be double-stranded or single-stranded. The deep rooting control gene may contain an additional sequence, such as a base sequence of an untranslated region (UTR), in addition to the ORF of the DRO1 gene or qSOR1 gene.

[0065] The method for obtaining (isolating) the deep-rootedness control gene is not particularly limited, but for example, a probe that specifically hybridizes with a portion of the base sequence of the deep-rootedness control gene may be prepared and a genomic DNA library or a cDNA library may be screened.

[0066] Another method for obtaining the deep-rootedness control gene is to use amplification techniques such as PCR. For example, primers are prepared from the 5' and 3' sequences (or their complementary sequences) of the cDNA of the deep-rootedness control gene, and these primers are used to perform PCR using genomic DNA (or cDNA) as a template, amplifying the DNA region between the two primers, thereby obtaining large quantities of DNA fragments containing the deep-rootedness control gene. Furthermore, chemically synthesized DNA can be produced, for example, using a commercially available oligonucleotide synthesizer.

[0067] Furthermore, whether or not an isolated candidate gene for a deep-root control gene has the desired deep-rootedness can be evaluated by observing whether deep-rootedness is induced by expression of the candidate gene in the plant from which it is derived.

[0068] The deep-rooting control gene can be used to elucidate the mechanism by which methane emissions are reduced 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 plants into which the deep-rooting control gene has been introduced, rice with deep-rooting traits can be obtained.

[0069] [Deep root regulatory protein] The deep-rootedness control protein is a translation product of the gene described in the "deep-rootedness control gene" column and has the activity of conferring the deep-rooted trait to rice. As described above, the deep-rootedness control protein does not induce the deep-rooted trait or inhibit the appearance of the trait.

[0070] Deep-rooted regulatory proteins may be isolated from natural sources or chemically synthesized. More specifically, the proteins include purified natural products, products of chemical synthesis procedures, and translation products produced by recombinant technology from prokaryotic or eukaryotic hosts, including, for example, bacterial cells, yeast cells, higher plant cells, insect cells, and mammalian cells.

[0071] That is, the deep-rootedness control protein can be obtained, for example, by inserting the deep-rootedness control gene into an appropriate expression vector, introducing the vector into appropriate cells, culturing the transformed cells to express the protein, and purifying it. The recombinant protein expressed in the host cell can be purified and recovered from the host cell or its culture supernatant by methods known to those skilled in the art. It is also possible to produce a transformed plant into which the deep-rootedness control gene has been introduced using the method described below, and then prepare the deep-rootedness control protein from the plant.

[0072] More specifically, 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 having 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 deep-rooted traits to rice; (c') A protein consisting of an amino acid sequence in which 25 or fewer amino acids have been substituted, deleted, added or inserted relative to the amino acid sequence set forth in SEQ ID NO: 1 or 2, and having the function of conferring deep-rooted traits to rice.

[0073] The protein (a') above is a protein encoded by the DRO1 gene or the qSOR1 gene, and has the activity of imparting the trait of deep rooting to rice.

[0074] With respect to the protein (b') above, 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 particularly preferably 96% or more, 97% or more, 98% or more, or 99% or more. For example, mutant proteins derived from rice are included in the category of the protein (b') above. These mutant proteins are proteins encoded by endogenous genes of rice.

[0075] With respect to the protein (c') above, 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, more preferably 1 to 20, 1 to 15, 1 to 10, or 1 to 5, and even more preferably 1 to 4, 1 to 3, or 1 to 2.

[0076] The deep-rooting regulatory protein is a polypeptide formed by peptide bonds between amino acids, but may also contain structures other than polypeptides, such as, but not limited to, sugar chains and isoprenoid groups.

[0077] [Expression vectors, cells, and transformants] Expression vectors incorporating the deep-rooting control gene, cells containing the expression vector or the deep-rooting control gene, and transformants into which the expression vector or the deep-rooting control gene has been introduced in an expressible manner can be used to produce rice plants containing the deep-rooting control gene in their genome. The expression vectors are used to produce deep-rooting control proteins. In other words, the expression vectors confer the deep-rooting trait to cells or individual organisms.

[0078] The type of vector used to construct the expression vector is not particularly limited, and can be selected from those capable of expression in host cells. That is, a promoter sequence can be selected appropriately depending on the type of host cell, and the promoter sequence and the deep-rooting control gene can be incorporated into, for example, a plasmid, phagemid, or cosmid, and used as the expression vector.

[0079] Host cells into which expression vectors can be introduced include, for example, bacterial cells, yeast cells, fungal cells other than yeast cells, and higher eukaryotic cells. Examples of bacterial cells include Escherichia coli cells. Examples of higher eukaryotic cells include plant cells and animal cells. Examples of plant cells include dicotyledonous plant cells and monocotyledonous plant cells. Examples of dicotyledonous plant cells include suspension culture cells of Solanaceae plants (e.g., tobacco BY-2 strain and tomato Sly-1 strain). Examples of monocotyledonous plant cells include the Oc strain, which is a suspension culture cell of rice. Examples of animal cells include insect cells, amphibian cells, reptile cells, avian cells, fish cells, and mammalian cells.

[0080] In the expression vector, the deep-rooted control gene is functionally linked to elements necessary for transcription (e.g., promoter, etc.). If necessary, enhancers, selection markers, splicing signals, poly(A) addition signals, and 5'-UTR sequences may also be linked. A promoter is a DNA sequence that exhibits transcriptional activity in host cells and can be selected appropriately depending on the type of host.

[0081] Examples of promoter sequences that can function in host cells include the 35S promoter of cauliflower mosaic virus, the nopaline synthase gene promoter of Agrobacterium, and the rice ubiquitin gene promoter. Furthermore, the promoter sequence of the deep-rootedness control gene may also be used as a recombinant expression promoter.

[0082] In the expression vector, the deep-rootedness control gene may be functionally linked to an appropriate terminator (e.g., the NOS terminator or the 35S terminator of the cauliflower mosaic virus) as needed. The type of appropriate terminator can be selected appropriately depending on the type of host cell, as long as it has a sequence that can terminate transcription of the gene transcribed by the above-mentioned promoter. Enhancers are used to increase the expression efficiency of the target gene, and examples include the omega sequence of the tobacco mosaic virus.

[0083] The expression vector may further contain a selection marker, such as a drug resistance gene for ampicillin, kanamycin, tetracycline, chloramphenicol, neomycin, hygromycin, or spectinomycin.

[0084] Furthermore, in the expression vector, the deep-rootedness control gene may be linked to a suitable tag sequence for protein purification or a suitable spacer sequence, if necessary.

[0085] Cells carrying a deep-rooting control gene in an expressible state can 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 and expressing a deep-rooting control protein. When using plant cells as hosts, plant-derived cells can be used and cultured as callus. Various methods known to those skilled in the art can be used to introduce an expression vector into a host cell. For example, an introduction method using calcium ions can be used to introduce an expression vector into E. coli cells, and an introduction method such as the polyethylene glycol method or electroporation can be used to introduce an expression vector into a plant cell.

[0086] The term "transformant" refers not only to cells, tissues, and organs into which the expression vector or deep-rooting control gene has been introduced in an expressible manner, but also to individual organisms. Such a transformant may be rice. The transformant may also be, for example, a microorganism such as Escherichia coli, an animal, or the like.

[0087] [Rice with deep-rooted traits] Rice plants having the trait of deep rooting are plants obtained by the production method described below. Rice plants having the trait of deep rooting have a deep rooting control gene.

[0088] As shown in the production method described below, rice with deep-rooted traits can be obtained by crossing rice with other rice varieties, and identifying the deep-rooted rice from the resulting plants and their progeny lines using the above-mentioned method for determining the degree of methane emission regulation in rice. Note that rice with deep-rooted traits that has been genetically engineered to contain a deep-rooted control gene is also included in the scope of the present invention.

[0089] [Method for producing rice with deep rooting traits] A method for producing rice plants with deep-rooted traits includes a step of carrying out a method for determining the degree of regulation of methane emission in rice plants according to one embodiment of the present invention and selecting rice plants with deep-rooted traits.

[0090] Therefore, the above description of the method for determining the degree of regulation of methane emission in rice is also applicable to the method for producing rice having deep-rooted traits.

[0091] The rice production method may further include a step of intraspecifically crossing rice having a deep-rooted trait with another rice, and in the selection step, rice having a deep-rooted trait may be selected from the rice obtained by the intraspecific crossing step or from rice of a progeny line.

[0092] In the crossing step, the rice having a deep-rooted trait used as a parent plant may be a rice having a deep-rooted trait according to one embodiment of the present invention. Furthermore, the rice having a deep-rooted trait used in the crossing step may be a rice having a deep-rooted trait selected by the method for determining the degree of regulation of methane emission in rice according to one embodiment of the present invention. That is, the rice production method may further include, prior to the crossing step, a selection step of selecting a rice having a deep-rooted trait from test rice plants by the method for determining the degree of regulation of methane emission in rice according to one embodiment of the present invention.

[0093] A method for producing rice plants having a deep-rooted trait according to another aspect of the present invention includes transfecting rice plants with any one of the following DNAs (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) a DNA encoding a protein having an amino acid sequence that has 90% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 or 2 and that has the function of conferring deep-rooted traits to rice; (c) DNA encoding a protein having an amino acid sequence in which 25 or fewer amino acids have been substituted, deleted, added, or inserted relative to the amino acid sequence set forth in SEQ ID NO: 1 or 2, and having the function of imparting deep-rooted traits to rice; The method includes introducing the vector into rice cells.

[0094] A preferred embodiment of the method for producing rice is a method for producing a transformed rice plant having a deep-rooted trait by the following steps (I) and (II): (I) introducing a deep-rooting control gene or an expression vector containing the gene into a plant cell; (II) A step of regenerating a plant body from the plant cell of step (I).

[0095] The step of introducing the deep root control gene (I) or an expression vector containing the gene into plant cells can be carried out by the method described in the above section [Expression vectors, cells, and transformants].

[0096] The regeneration of plants from plant cells as described in (II) above can be carried out by methods known to those skilled in the art depending on the type of plant, including, but not limited to, the method described by Fujimura et al. (Fujimura et al., Tissue Culture Lett. 2, 1995, 74).

[0097] Whether or not the regenerated plant has the trait of deep rooting can be determined by a method for determining the degree of regulation of methane emission in rice according to one embodiment of the present invention.

[0098] In the rice production method, once a transformed plant is obtained in which the deep-rooting control gene of the present invention has been introduced into its chromosome, it is possible to obtain offspring from the plant through sexual or asexual reproduction. Furthermore, cells and propagation materials (e.g., seeds, fruits, cuttings, stalks, roots, stocks, callus, protoplasts, etc.) can be isolated from the plant, its progeny, or clones, and used to mass-produce the plant. Furthermore, these plant cells, plants containing the cells, progeny and clones of the plant, and propagation materials of the plant, its progeny, and clones can be used to impart the deep-rooting trait to plants.

[0099] According to the present invention, it is possible to contribute to the mitigation of climate change by reducing methane emissions, thereby contributing to the achievement of the Sustainable Development Goals (SDGs).

[0100] The present invention is not limited to the above-described embodiments, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention. [Example]

[0101] [1. Rice with deep or shallow rooting characteristics]

[0102] Figure 1 shows the difference in root elongation angle due to the DRO1 gene and / or qSOR1 gene. The root elongation angle varied depending on the DRO1 gene and / or qSOR1 gene, as shown in Figure 1. Specifically, lines in which both genes were functional were deep-rooted, while lines with homozygous loss-of-function DRO1 and loss-of-function qSOR1 genes were ultra-shallow-rooted. Lines in which one gene was functional exhibited intermediate root angles, with DRO1(+) / qSOR1(-) being deeper-rooted than DRO1(-) / qSOR1(+). Here, DRO1(-) / qSOR1(+) is the allele inherent to IR64 and was considered the control in this example. The four lines described in Figure 3 of Reference 2 were used as these four lines.

[0103] [2. Rice cultivation] Germinated seeds of the four strains were grown in seedling boxes until the fifth leaf developed, then transplanted into puddled paddy fields with three plants per plant. The planting density was 30cm (row spacing) x 15cm (row spacing), with each plot consisting of 4 rows x 8 plants, and the cultivation experiment was carried out with four replications. The experiment was carried out in the paddy fields of the National Agriculture and Food Research Organization's Agricultural Environment Research Division in Tsukuba City, Ibaraki Prefecture, and the plants were grown under permanently flooded conditions until harvest.

[0104] [3. Measuring methane emissions] Methane emissions were measured using a closed chamber method (Minamikawa et al. 2015. ISBN:978-4-931508-16-3 (online)). An acrylic chamber with a base area of ​​30 cm x 60 cm, equivalent to the area of ​​four plants, was used, and the amount of methane accumulated in the chamber was analyzed using a gas chromatograph with an FID detector to measure the amount of methane emitted per unit time and unit area (flux).

[0105] The results are shown in Figures 2 and 3. Figure 2 shows the time course of methane emissions from four rice lines with different DRO1 / qSOR1 alleles. The numbers at the bottom of the figure represent the percentage change in the deep-rooted lines (DRO1(+) / qSOR1(+)) compared to IR64 (DRO1(-) / qSOR1(+)). Figure 3 shows the difference in cumulative methane emissions between lines over the entire measurement period. The numbers at the top of the figure represent the percentage change in emissions from each line compared to IR64 (DRO1(-) / qSOR1(+)). Deep-rooted lines bred from IR64, carrying functional alleles for DRO1 and qSOR1, exhibited reduced methane emissions during the latter half of growth (Figure 2), and their overall emissions were 14% lower than those of IR64 (Figure 3). Conversely, methane emissions increased in lines with shallower rooting than IR64 (DRO1(-), qSOR1(-)) (Figures 2 and 3).

[0106] [4. Relationship between methane emissions and yield] Six mature rice plants were harvested from each plot, and the yield (polished rice weight) was investigated. The polished rice was obtained by hulling the threshed rice in salt water at a specific gravity of 1.06 and then hulling the sunk seed rice. The weight was calculated based on a moisture content of 15%.

[0107] The results are shown in Figures 4 and 5. Figure 4 shows the milled rice yield (15% moisture) of four lines with different DRO1 / qSOR1 genes. The numbers on the figure represent the percentage change for each line compared to IR64. Figure 5 shows the methane emissions per unit yield. The numbers on the figure represent the percentage change for each line compared to IR64. The deep-rooted line (DRO1(+) / qSOR1(+)) had a yield equivalent to that of IR64 (+0.3%, Figure 4), so methane emissions were suppressed (-15%, Figure 5) while maintaining yield. The line (DRO1(+), qSOR1(-)), which is slightly deeper-rooted than IR64, showed no change in methane emissions per unit area (Figure 3), but increased yield (+19.7%, Figure 4), resulting in a decrease in methane emissions per unit yield (Figure 5).

[0108] [5. Measurement of iron(II) concentration in soil water] As soil becomes more reductive, methane production by microorganisms increases, while in the oxidative areas around the roots, methane decomposition by methane-oxidizing bacteria occurs. Therefore, soil water from 1 to 11 cm deep was collected through a porous cup (10 cm), and the dissolved ferrous iron concentration in the soil water, an indicator of redox, was analyzed using an ICP spectrometer.

[0109] The results are shown in Figure 6. Figure 6 shows the changes in ferrous iron (Fe(II)) concentrations over time in soil water for four lines with different DRO1 / qSOR1 genes. In the deep-rooted line (DRO1(+) / qSOR1(+)), ferrous iron concentrations were kept lower throughout the growth period compared to IR64. This suggests that the deep rooting of the lines results in a relatively oxidized soil, which suppresses methane production by microorganisms and promotes methane oxidation around the oxidized rice roots, leading to reduced methane emissions (Figure 6). [Industrial Applicability]

[0110] The present invention can be used in the agricultural field, and also in the environmental field through the maintenance and development of agriculture.

Claims

1. A process for selecting rice having a deep-rooted trait with a deep root elongation angle; Growing the selected rice plant; Inclusive of In the selection step, a rice plant having in its genome a deep-rootedness control gene encoding a protein having an activity of imparting a deep-rootedness trait is selected; The deep rooting control gene is a DNA selected from the group consisting of any one of the following (a) to (c): (a) a DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 or 2; (b) a DNA encoding a protein having 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 an activity of imparting a deep-rooted trait to rice; (c) a DNA encoding a protein having an amino acid sequence in which 25 or less amino acids have been substituted, deleted, added or inserted relative to the amino acid sequence set forth in SEQ ID NO: 1 or 2 and having an activity of imparting deep-rooted traits to rice; Including, The deep-rooted trait is a property in which the roots, which are underground organs of a plant body, extend into the ground at a deep angle relative to the ground surface. The deep angle indicates that, when the ground surface is horizontal and the vertical downward angle is 90 degrees, the proportion of roots with root growth angles of at least 50 degrees or more out of the total number of roots is 50% or more.

2. 2. The method for reducing methane emissions from paddy fields as described in claim 1, wherein in the selecting step, a methane emission reduction management database is obtained that contains information on the amount of methane emission reduction during cultivation of rice having deep-rooted traits compared to the amount of methane emission from a reference paddy field, for each rice variety, and the rice to be grown is selected by referring to the methane emission reduction management database.

3. 3. The method for reducing methane emissions from paddy fields according to claim 2, further comprising a step of calculating an amount of methane emission reduction from the rice to be grown by referring to the information on the amount of methane emission reduction, and converting the calculated amount of methane emission reduction into carbon credits.

4. A method for determining the degree of regulation of methane emission in rice, comprising: The method includes a step of determining the degree of regulation of methane emission from a test rice plant by using as an index whether or not the test rice plant has a deep-rooted trait, that is, a deep root growth angle; In the step of determining, the degree of regulation of methane emission from the test rice is determined based on whether or not the test rice has a deep-rootedness control gene in its genome that encodes a protein having an activity that confers a deep-rootedness trait; The deep rooting control gene is a DNA selected from the group consisting of any one of the following (a) to (c): (a) a DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 or 2; (b) a DNA encoding a protein having 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 deep-rooted traits to rice; (c) a DNA encoding a protein having an amino acid sequence in which 25 or less amino acids have been substituted, deleted, added, or inserted relative to the amino acid sequence set forth in SEQ ID NO: 1 or 2 and having the function of imparting deep-rooted characteristics to rice; Including, The deep-rooted trait is a property in which the roots, which are underground organs of a plant body, extend into the ground at a deep angle relative to the ground surface. The deep angle indicates that the percentage of roots with a root growth angle of at least 50 degrees or more is 50% or more of the total number of roots when the ground surface is horizontal and the vertical downward angle is 90 degrees.

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