Method for reducing methane emissions from paddy fields, method for determining the degree of control of methane emissions in rice, and rice packaging

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 emission from paddy fields, a method for determining the degree of regulation of methane emission in rice, and a packaged product of rice. [Background technology]

[0002] Since the Paris Agreement came into force in 2016, efforts towards decarbonization and global warming prevention have been accelerating worldwide. As part of this trend towards 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 main 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 to develop methane reduction technology that can be easily adopted by ordinary farmers.

[0003] As a method for reducing methane emissions from paddy fields, a method of making paddy field soil aerobic through water management is known. Non-Patent Document 1 describes reducing methane emissions from paddy fields by extending mid-season drainage. Non-Patent Document 2 describes a method for reducing methane emissions from paddy fields 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. Examples of such burdens include additional water management work and psychological stress during water shortages. In addition, the success or failure of water management is affected by the weather and the drainage properties of paddy fields, which makes the effect of reducing methane emissions unstable. Furthermore, when establishing a subsidy system for producers who contribute to methane reduction, it is difficult to verify whether the producers actually performed the management, making it difficult to fairly award rewards according to the reduction in methane emissions. The above points hinder the spread 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 rice by using as an indicator whether or not the test rice has a deep-rooted trait.

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

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

[0011] [Figure 1] FIG. 1 shows differences in rice root conditions due to the DRO1 gene and the qSOR1 gene. [Diagram 2] 1 is a graph showing methane emission amounts for each rice plant in the examples. [Diagram 3] 1 is a graph showing the cumulative methane emission amount for each rice plant in the examples. [Figure 4] 1 is a graph showing the milled rice yield of each rice plant in the examples. [Diagram 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 ferrous iron concentration in soil water for each rice plant in the examples. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

[0013] [Definition of terms, etc.] In this specification, "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. In this specification, "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 cultivated under flooding, and can also be said as the amount of methane gas generated when rice is cultivated under flooding. "Methane emission" can also be 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 cultivars 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 be rephrased 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 a step of selecting rice plants having deep-rooted characteristics and a step of growing the selected rice plants. This allows reduction of methane emissions from paddy fields to be realized. The present inventors have found that when rice plants having deep-rooted characteristics are cultivated, the amount of methane emissions from paddy fields is reduced compared to when rice plants not having deep-rooted characteristics are cultivated, and have completed the present invention based on this new finding. Here, the "deep-rooted characteristics" refers to the property that the roots, which are underground organs of the plant body, extend into the ground at a deep angle to the ground surface. In addition, the "deep angle" means that the ratio of the number of roots whose angle with respect to the ground surface is at least 50 degrees is 50% or more, preferably 60% or more, more preferably 70%, and even more preferably 80% or more of the total number of roots.

[0018] In the selecting step, a variety exhibiting a deep-rooted phenotype is selected from among the rice varieties. In the selecting step, a rice variety may be selected based on whether the roots grow at a deeper angle than a certain rice variety as a reference. In the selecting 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 the amount of methane emissions from paddy fields compared to the cultivation of rice that does not have the deep-rooted trait. Here, the concept of methane emission amount includes the degree of methane emission, which indicates whether the methane emission amount of a certain rice plant individual is relatively high or low compared to other rice plants.

[0020] Methane gas is attracting attention as a reduction target for realizing decarbonization and global warming prevention. Paddy fields where rice is cultivated are one of the main sources of methane emissions. Therefore, technology that can efficiently and easily reduce methane emissions from paddy fields is desired. According to the method for reducing methane emissions from paddy fields, by selecting individuals with deep root traits in rice, individuals with traits that reduce methane emissions can be selected. Therefore, methane emissions from paddy fields can be reduced simply by selecting and cultivating individuals with traits that reduce methane emissions. In addition, since methane emissions from paddy fields can be reduced simply by cultivating individuals selected in the method for reducing methane emissions from paddy fields, the reduction effect can be obtained stably, and it is easy to verify whether the reduction measures have been implemented.

[0021] In the selection step, rice having in its genome a deep-rootedness control gene encoding a protein having activity to impart a deep-rooted trait may be selected. By selecting and growing rice having in its genome a deep-rootedness control gene, it is possible to reduce methane emissions from paddy fields. In other words, the deep-rootedness control gene may be a gene involved in determining the amount of methane emissions (methane emission control gene). In the rice selection step, as an example, a rice variety that is known in advance to have a deep-rootedness control gene in its genome may be selected.

[0022] The deep rooting control gene is a DNA represented by 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 a deep-rooted trait to rice; It is a gene containing

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

[0024] In the step of selecting rice, it is possible to determine whether rice, which is not known whether it has a deep-rootedness control gene in its genome, has the deep-rootedness control gene in its genome, and select rice having the deep-rootedness control gene in its genome based on the determination result. In the step of selecting rice, an example of a method for determining whether rice has a deep-rootedness control gene in its genome is a 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 one 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 having a deep-rooted trait, methane emissions from the paddy field are reduced.

[0026] In addition, in the method for reducing methane emissions from paddy fields, 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 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 may be selected by referring to the methane emission reduction management database.

[0027] Such a database may be constructed in advance and made public 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 having deep-rooted traits, rice producers can obtain carbon credits as compensation for the reduction in methane emissions, and can earn profits by trading the obtained carbon credits in the emissions trading market. That is, the method for reducing methane emissions from paddy fields may further include a step 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] Here, carbon credits as compensation for methane emission reductions are granted by a carbon credit certification body and can be traded in an emissions trading market approved by the certification body. As a result, producers of rice with deep-rooted traits can trade the carbon credits obtained by cultivating the rice, thereby gaining added value equivalent to the carbon credits. In addition, sellers of rice with deep-rooted traits can add the above-mentioned added value to the selling price of the rice.

[0029] Rice producers can reduce methane emissions from rice cultivation by selecting the rice they will grow 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 reduced in rice. The determination method determines whether rice has a deep-rooted trait or not, thereby determining whether methane emission is reduced or not reduced.

[0031] A method for determining an aspect of the present invention 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. In the determining step, it is determined whether or not the test rice plant exhibits a deep-rooted phenotype. In the determining step, the degree of regulation of methane emission from 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 standard rice variety. In the determining step, the degree of regulation of methane emission from the rice plant may also be determined based on the angle of the roots compared to that of the standard rice variety.

[0032] The test rice in the determination method may be a rice plant whose deep-rooted trait is unknown. The test rice may be a candidate plant for breeding material or a plant obtained by a breeding process. Candidate plants for breeding material include, for example, parent plants used for crossbreeding and plants used for molecular breeding using recombinant gene technology. Plants obtained by a breeding process include, for example, plants obtained by intraspecific hybridization of rice and their progeny lines. The rice may also be a plant obtained by intervarietal hybridization, such as a hybrid plant between rice belonging to a certain variety and rice belonging to another variety, and its progeny line. The rice may also be a plant obtained by hybridization between varieties known to have deep-rooted traits, and its progeny line.

[0033] In addition, the rice may be a plant obtained by crossing a variety known to have a deep-rooted trait with a variety unknown to have the deep-rooted trait, and its progeny. In addition, the rice may be a plant obtained by crossing varieties unknown to have the deep-rooted trait, and its progeny. In addition, the rice may be a plant obtained by crossing a variety known to have a deep-rooted trait with a variety known not to have the deep-rooted trait, and its progeny. In addition, the rice may be a plant obtained by crossing individuals known to have the deep-rooted trait with each other, and its progeny.

[0034] In the present specification, a 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 judgment step, the degree of regulation of methane emission of the test rice may be judged by using as an index whether or not the test rice has a deep-rootedness control gene in its genome that encodes a protein having activity that confers the trait of deep rootedness. In the judgment step, as an example, the genotype of the deep-rootedness control gene present in the genome of the test rice is judged to judge the rice based on its deep rootedness.

[0036] The method for determining the presence or absence of a DNA selected from the group consisting of DNA of any one of the following (a) to (c) is carried out in a test rice plant: (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 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 the function of imparting a deep-rooted trait to rice; The method may include a step of testing whether or not a deep-rootedness control gene comprising the above-mentioned component (a) or a protein encoded by the deep-rootedness control 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] A DNA sample from the test rice can be prepared by a method 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 to be subjected to 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, ears, leaves, roots, stems, seeds, endosperm, bran, embryos, etc., but is not limited thereto.

[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, the determination method amplifies the region in the DNA of the test rice plant using a primer set that amplifies the 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 the 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 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 length of the genomic DNA may be amplified, or an ORF region (e.g., the DNA region shown in SEQ ID NO: 3 or 4) or a part of the ORF region encoding the deep-rootedness control protein may be amplified.

[0042] The region to be amplified may be a region containing at least a molecular marker for testing the presence or absence of a mutation in the deep-rootedness control gene. Examples of the molecular marker include SNP markers, AFLP (amplified fragment length polymorphism) markers, RFLP markers, microsatellite markers, SCAR markers, and CAPS markers.

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

[0044] Amplification of the region in the DNA of the test rice plant can be carried out 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 an isotope of P, 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 having different wavelengths (e.g., NED, 6-FAM, VIC, PET) may be mixed.

[0047] The PCR reaction conditions can be appropriately set depending on the type of DNA polymerase and PCR device used, the length of the amplified fragment, and the like. As cycle conditions, a three-step PCR method in which three steps, a denaturation step, an annealing step, and an extension step, are one cycle, and a two-step PCR method in which two steps, a denaturation step, an annealing step, and an extension step, are one cycle, can be applied. An example of the PCR reaction conditions is 90 to 100°C for 40 to 60 seconds (for example, 95°C for 50 seconds), followed by 90 to 100°C (for example, 95°C) for 5 seconds, annealing for 10 to 20 seconds (for example, 15 seconds), and 65 to 80°C for 10 to 30 seconds (for example, 72°C for 20 seconds), for a total of 30 to 60 cycles (for example, 40 cycles). The annealing temperature can be reduced stepwise every predetermined cycle from an initial annealing temperature of 60 to 70°C (for example, 66°C) to a final annealing temperature of 50 to 60°C (for example, 56°C). 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 (registered trademark)-PCR method and Tm-shift genotyping method (Fukuoka et al., Breed Sci 58:461-464, 2008) may be used. That is, TaqMan (registered trademark) probe may be used. By using real-time PCR method, a high-throughput discrimination method can be provided.

[0049] Comparison of the molecular weight or base sequence of the amplified DNA fragments can be performed by a method known to those skilled in the art, such as DNA sequencing, such as the Sanger method. For example, the DNA fragments can be denatured by heating or the like, and electrophoresed 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. The amplified DNA fragments may also be analyzed by determining the base sequence using an automatic DNA sequencer or the like.

[0050] When labeled DNA is used, the mobility of DNA fragments after electrophoresis can be detected and analyzed by autoradiography using 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 a silver staining method or the like. For example, DNA fragments can be amplified from a variety having a deep-rooted trait and a test rice plant using primers, and the molecular weights can be compared to determine whether the test rice plant has a deep-rooted trait. If the molecular weights match, the test rice plant is determined to have a 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 having the deep-rooting trait to determine 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 base sequence of both allele genes match those of a plant with a deep-rooted trait, or that the amino acid sequence matches those of the allele. Therefore, if the molecular weight, base sequence, or amino acid sequence of one allele gene is different from that of a plant with a deep-rooted trait, but the other is the same as that of a plant with a deep-rooted trait, it is not included in "matching."

[0053] The method of determination may be to test for the presence or absence of a mutation in the deep-rootedness control gene using a molecular marker consisting of a continuous DNA containing the 354th base of the DNA consisting of the base sequence shown in SEQ ID NO: 3 or the base corresponding to the 217th base in the base sequence of SEQ ID NO: 4 (SNP). 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 having a trait of reducing methane emissions. The packaged product is a package of multiple rice plants determined to have deep roots 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 roots control gene that encodes a protein having activity that imparts a deep roots trait. The rice contained in the packaged product may be, for example, a seedling, a seed, or the like. The packaged product may state that the rice contained in the packaged product has a trait of reducing methane emissions. A rice producer can achieve a reduction in methane emissions in rice cultivation simply by obtaining the packaged product and cultivating the rice contained in the packaged product.

[0055] [Deep root control gene] The deep-rootedness control gene encodes a protein that has the activity of imparting the deep-rooted trait to rice (deep-rootedness control activity). Here, the "deep-rootedness trait" refers to the property of the roots, which are underground organs of the plant body, growing underground at a deep angle to the ground surface. In addition, the "deep angle" means that the proportion of roots with an angle of at least 50 degrees to the ground 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 a deep-rooted trait, the plant has deeper roots than a plant in which the activity is reduced or inhibited, and emits less methane than a plant in which the activity is reduced or inhibited. Also, when the activity of the protein is reduced or inhibited, the plant has shallower roots than a plant in which the activity is not reduced or inhibited, and emits more methane than a plant 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 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 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 the function of conferring deep-rooted traits to rice.

[0058] Among the DNAs in (a) above, the DNA encoding a 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 cultivar Kinandang Patong, and is a DNA encoding a protein having 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 is a DNA that 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 characteristics to rice.

[0060] Examples of genes having an activity of imparting deep-rooted traits to rice include the DRO1 gene and the qSOR1 gene. It is described in References 1 and 2 that these genes have an activity of imparting 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 an activity of imparting deep-rooted traits, have an activity of imparting 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 endogeneous 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, 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 be 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 include an additional sequence, such as a base sequence of an untranslated region (UTR), together with 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 can be prepared and a genomic DNA library or cDNA library can be screened.

[0066] Another method for obtaining the deep-rootedness control gene is to use an amplification method 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 or the like using genomic DNA (or cDNA) or the like as a template to amplify the DNA region sandwiched between the two primers, thereby obtaining a large amount of DNA fragments containing the deep-rootedness control gene. Furthermore, chemically synthesized DNA can be produced, for example, by a commercially available oligonucleotide synthesizer.

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

[0068] The deep-rootedness control gene can be used to elucidate the mechanism by which methane emissions are reduced in rice. The deep-rootedness control gene can also be used to produce a transformant by introducing the gene into a rice plant or cell, for example by incorporating the gene into an expression vector. Rice with deep-rooted characteristics can be obtained by cultivating rice into which the deep-rootedness control gene has been introduced.

[0069] [Deep root control protein] The deep-rootedness control protein is a translation product of the gene described in the deep-rootedness control gene column above, and has an activity of imparting deep-rootedness to rice. As described above, the deep-rootedness control protein does not cause the deep-rootedness trait to appear, nor does it inhibit the appearance of the trait.

[0070] The deep root control protein may be isolated from a natural source or may be chemically synthesized. More specifically, the protein includes natural purified 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-rooted control protein can be obtained, for example, by inserting the deep-rooted control gene into an appropriate expression vector, introducing the vector into an appropriate cell, culturing the transformed cell 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 a method known to those skilled in the art. It is also possible to prepare a transformed plant body into which the deep-rooted control gene has been introduced, and prepare the deep-rooted control protein from the plant body, using the method described below.

[0072] More specifically, the deep root control protein is any one of the following proteins (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 a deep-rooted trait 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 above protein (a') is a protein encoded by the DRO1 gene or the qSOR1 gene, and has the activity of imparting the deep-rooted trait to rice.

[0074] Regarding 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-rooted control protein is a polypeptide formed by peptide bonds between amino acids, but may also contain structures other than polypeptides. Examples of structures other than polypeptides include, but are not limited to, sugar chains and isoprenoid groups.

[0077] [Expression vectors, cells, and transformants] An expression vector incorporating the deep-rootedness control gene, a cell containing the expression vector or the deep-rootedness control gene, and a transformant into which the expression vector or the deep-rootedness control gene has been introduced so as to be expressible can be used to produce rice having the deep-rootedness control gene in its genome. The expression vector is used to produce a deep-rootedness control protein. In other words, the expression vector confers the deep-rootedness trait to a cell or an individual organism.

[0078] The type of vector for constructing the expression vector is not particularly limited, and one that can be expressed in the host cell may be appropriately selected. That is, a promoter sequence may be appropriately selected according to the type of the host cell, and the promoter sequence and the deep-rooted control gene may be incorporated into, for example, a plasmid, a phagemid, or a cosmid, and used as the expression vector.

[0079] Examples of host cells into which an expression vector is introduced include 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 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.). In addition, an enhancer, a selection marker, a splicing signal, a polyA addition signal, a 5'-UTR sequence, etc. may be linked as necessary. The promoter is a DNA sequence that exhibits transcription activity in a host cell, and can be appropriately selected depending on the type of host.

[0081] Examples of promoter sequences operable in a host cell include the 35S promoter of the cauliflower mosaic virus, the nopaline synthase gene promoter of Agrobacterium, and the rice ubiquitin gene promoter. In addition, the sequence of the promoter region in the deep-root control gene may be used as a recombinant expression promoter.

[0082] In the expression vector, the deep-rooted control gene may be functionally linked to an appropriate terminator (e.g., NOS terminator and 35S terminator of cauliflower mosaic virus) as necessary. The type of appropriate terminator may be appropriately selected depending on the type of host cell, and may be any sequence that can terminate the transcription of the gene transcribed by the above-mentioned promoter. Enhancers are used to increase the expression efficiency of the target gene, and examples of such enhancers include the omega sequence of 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] The cells carrying the deep-rooted control gene in an expressible state may be cells into which an expression vector incorporating the deep-rooted control gene has been introduced, and can be used as a production system for manufacturing and expressing the deep-rooted control protein. When using plant cells as the host, 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 the host cell. For example, an introduction method using calcium ions can be used to introduce the expression vector into E. coli cells, and an introduction method such as the polyethylene glycol method or electroporation can be used to introduce the expression vector into the plant cell.

[0086] The term "transformant" refers to a cell, tissue, or organ into which the expression vector or the deep-root control gene is introduced so as to be capable of expression, and also includes an individual organism. 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 having a deep-rooted trait is a plant obtained by the production method described below. Rice having a deep-rooted trait has a deep-rootedness controlling gene.

[0088] As shown in the production method described below, rice having the deep-rooted trait can be obtained by crossing rice having the deep-rooted trait with another rice, and determining from the resulting plant and its progeny by the above-mentioned [method for determining the degree of regulation of methane emission in rice]. Note that rice having the deep-rooted trait that has been genetically engineered to have a deep-rooted control gene is also included in the scope of the present invention.

[0089] [Method for producing rice with deep-rooted traits] A method for producing rice having a deep-rooted trait includes a step of carrying out a 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-rooted trait.

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

[0091] The rice production method may further include a step of intraspecific hybridizing 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 hybridization step or from a progeny of the rice.

[0092] In the crossing step, the rice having a deep-rooted trait used as a parent plant may be the rice having a deep-rooted trait according to one embodiment of the present invention. In addition, the rice having a deep-rooted trait used in the crossing step may be 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 a selection step of selecting 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, prior to the crossing step.

[0093] A method for producing rice having a deep-rooted trait according to another aspect of the present invention comprises transfecting rice with any one of the following DNAs (a) to (c) or an expression vector containing the DNA: (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 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 the function of imparting a deep-rooted trait to rice; The method includes introducing the vector into a rice cell.

[0094] A preferred embodiment of the method for producing rice includes a method for producing a transformed rice plant having a deep-rooted trait by the following steps (I) and (II): (I) introducing a deep-rootedness 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 a plant cell can be carried out by the method described in the above section [Expression vectors, cells, and transformants].

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

[0097] Whether or not the regenerated plant body has a deep-rooted trait 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, if a transformed plant body having the deep-rooting control gene of the present invention introduced into its chromosome is obtained, it is possible to obtain offspring from the plant body by sexual or asexual reproduction. It is also possible to isolate cells or propagation materials (e.g., seeds, fruits, cuttings, stalks, roots, stocks, calli, protoplasts, etc.) from the plant body or its progeny or clones, and mass-produce the plant body based on them. These plant cells, plants containing the cells, progeny and clones of the plant body, and propagation materials of the plant body, 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, which can contribute 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. EXAMPLES

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

[0102] FIG. 1 shows the difference in root elongation angle due to the DRO1 gene and / or qSOR1 gene. The root elongation angle differed depending on the DRO1 gene and / or qSOR1 gene as shown in FIG. 1. That is, the line in which both genes are functional became a deep-rooted type, and the line having homozygous functional loss-of-function DRO1 and functional loss-of-function qSOR1 genes became an ultra-shallow-rooted type. The line in which one is functional became an intermediate angle, and among them, DRO1(+) / qSOR1(-) was a deeper-rooted type than DRO1(-) / qSOR1(+). Here, DRO1(-) / qSOR1(+) is an allele that IR64 originally has, and was considered as a control in this example. As these four lines, the four lines described in FIG. 3 of Reference 2 were used.

[0103] [2. Rice Cultivation] The germinated rice seeds of the above four lines were grown in seedling boxes until the fifth leaf developed, and then transplanted into puddled paddy fields with three plants per plant. The planting density was 30cm (row spacing) x 15cm (plant spacing), and each plot had a colony of 4 rows x 8 plants, with four replicates of the cultivation test. The test 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 cultivated under permanent flooding conditions until harvest.

[0104] 3. Measurement of 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 bottom area of ​​30cm x 60cm, equivalent to the area of ​​four plants, was used, and the amount of methane accumulated in the chamber was analyzed by a gas chromatograph equipped 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 genes, and the numbers at the bottom of the figure indicate the percentage change in the deep-rooted lines (DRO1(+) / qSOR1(+)) compared to IR64 (DRO1(-) / qSOR1(+)). Figure 3 shows the difference between the lines in the cumulative methane emissions over the entire measurement period, and the numbers on the top of the figure indicate the percentage change in emissions from each line compared to IR64 (DRO1(-) / qSOR1(+)). The deep-rooted lines with functional alleles of DRO1 and qSOR1 bred from the rice cultivar IR64 showed a decrease in methane emissions in the latter half of growth (Figure 2), and the emissions throughout the growth period were 14% lower than those of IR64 (Figure 3). Conversely, methane emissions increased in lines with shallower roots 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. Polished rice was obtained by selecting hulled rice in salt water at a specific gravity of 1.06, and hulling the sunken rice seeds. 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, and the numbers on the figure indicate the change (%) of each line compared to IR64. Figure 5 shows the methane emission per yield, and the numbers on the figure indicate the change (%) of each line compared to IR64. The yield of the deep-rooted line (DRO1(+) / qSOR1(+)) was equivalent to that of IR64 (+0.3%, Figure 4), so methane emission was suppressed while maintaining the yield (-15%, Figure 5). Note that the line (DRO1(+), qSOR1(-)), which is slightly deeper rooted than IR64, did not change in methane emission per unit area (Figure 3), but the yield increased (+19.7%, Figure 4), so methane emission per yield decreased (Figure 5).

[0108] [5. Measurement of ferrous iron 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, which is an indicator of redox, was analyzed using an ICP luminescence device.

[0109] The results are shown in Figure 6. Figure 6 shows the changes in ferrous iron (Fe(II)) concentration over time in soil water for four strains with different DRO1 / qSOR1 genes. In the deep-rooted strain (DRO1(+) / qSOR1(+)), ferrous iron concentration was kept lower throughout the growth period compared to IR64. In other words, it was thought that the deep-rooted traits created a relatively oxidative state in the soil, suppressing methane production by microorganisms and promoting methane oxidation around the oxidative rice roots, which led to a reduction in methane emissions (Figure 6). [Industrial Applicability]

[0110] The present invention can be used in the agricultural field, and can also be used 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.

Images