Plant with increased gamma-aminobutyric acid content and method for producing same

Abstract

The purpose of the present invention is to provide a method for producing a plant having an increased gamma-aminobutyric acid (GABA) content, without incidental sterility and stunting, and to provide said plant. The inventors discovered that introducing a mutation into a regulatory sequence upstream of the GABA-T gene in a plant, thereby suppressing the expression level of said gene, can increase the GABA content without causing sterility or stunting.

Description

Plants with improved gamma-aminobutyric acid content and manufacturing method thereof

[0001] The present invention relates to a plant having an improved concentration of γ-aminobutyric acid (GABA) and a method for producing the same.

[0002] GABA is an amino acid that functions as a neurotransmitter in the human central nervous system, and its oral ingestion is also known to be effective in suppressing elevated blood pressure, relieving stress, improving sleep quality, maintaining skin elasticity, etc. Therefore, there is a high demand for plants with high GABA content, such as tomatoes, and there is a need to develop technology to further increase the amount of GABA accumulated in plants.

[0003] For example, enhancing GABA biosynthesis or inhibiting GABA degradation is thought to be effective methods for increasing the amount of GABA accumulated in tomato fruits. Methods for enhancing GABA biosynthesis include overexpressing the glutamic acid decarboxylase (GAD) gene involved in GABA biosynthesis using recombinant technology (Non-Patent Documents 1 and 2), and using genome editing to remove the autoinhibitory domain of the GAD protein and enhance its activity (Non-Patent Document 3). Meanwhile, a method for inhibiting GABA degradation has been reported in which the expression of the GABA-degrading enzyme (γ-aminobutyric acid transaminase: GABA-T) is inhibited using RNA interference (RNAi) (Non-Patent Document 4).

[0004] However, although the suppression of GABA-T gene expression described in Non-Patent Document 4 was effective in increasing GABA accumulation in tomato fruits, it has been reported to result in poor traits such as sterility and dwarfism. Therefore, the approach of suppressing GABA degradation has not yet been utilized in breeding plants with high GABA content.

[0005] Takayama et al. 2015 Plant Cell Physiol 56, 1533-1545 Takayama et al. 2017 Plant Cell Rep 36, 103-116 Nonaka et al. 2017 Sci Rep 7.1, 7057Koike et al. 2013 Plant Cell Physiol 54, 793-807

[0006] The present invention has been made in consideration of the problems associated with the above-mentioned prior art, and aims to provide a method for producing a plant with an improved GABA concentration that does not result in sterility or dwarfism, and the plant.

[0007] As a result of intensive research to achieve the above-mentioned object, the present inventors have found that the GABA content in plants can be increased by introducing a mutation into the regulatory sequence upstream of the GABA-T gene and suppressing the expression level of the gene. Meanwhile, they have demonstrated that the sterility and dwarfism reported in the degradation of GABA-T mRNA by RNAi (Non-Patent Document 4) do not occur when a mutation is introduced into the regulatory sequence, leading to the completion of the present invention.

[0008] That is, the present invention provides the following aspects.

[0009] [1] A method for producing a plant having an improved concentration of gamma-aminobutyric acid (GABA), comprising: (1) a step of introducing a mutation into a regulatory sequence located upstream of a gamma-aminobutyric acid aminotransferase (GABA-T) gene in a plant cell to suppress expression of the gene; and (2) a step of regenerating a plant from the plant cell in which expression of the gene has been suppressed in step (1).

[0010] [2] The method according to [1], wherein the plant cell is a tomato cell, and the mutation is introduced into the DNA sequence of SEQ ID NO: 1 or a sequence corresponding thereto in the regulatory sequence.

[0011] [3] The method according to [1], wherein the plant cell is a tomato cell, and the mutation is introduced into a DNA sequence consisting of positions 2056 to 2422 of SEQ ID NO: 1 in the regulatory sequence, or a sequence corresponding thereto.

[0012] [4] The production method according to any one of [1] to [3], wherein the plant cell is a cell in which the function of the C-terminal autoinhibitory domain of glutamic acid decarboxylase (GAD) has been lost.

[0013] [5] (3) The production method according to any one of [1] to [3], further comprising a step of crossing the plant obtained in the step (2) with a plant in which the function of the C-terminal autoinhibitory domain in GAD has been lost.

[0014] [6] A method for producing an edible part having an improved GABA concentration, comprising the steps of producing a plant body having an improved GABA concentration by the method according to any one of [1] to [5], and obtaining an edible part from the plant body.

[0015] [7] A plant having an improved GABA concentration, in which a mutation has been artificially introduced into a regulatory sequence located upstream of the GABA-T gene, thereby suppressing the expression of the gene.

[0016] [8] The plant according to [7], which is a tomato plant, into which the mutation has been introduced into the DNA sequence set forth in SEQ ID NO: 1 or a sequence corresponding thereto in the regulatory sequence.

[0017] [9] The plant according to [7], which is a tomato plant, into which the mutation has been introduced into a DNA sequence consisting of positions 2056 to 2422 of SEQ ID NO: 1 in the regulatory sequence, or a sequence corresponding thereto.

[0018]

[10] The plant according to any one of [7] to [9], further comprising a loss of function of the C-terminal autoinhibitory domain in GAD.

[0019]

[11] An edible portion of the plant body according to any one of [7] to

[10] .

[0020] According to the present invention, it is possible to provide a method for producing a plant having an improved GABA concentration without causing sterility or dwarfism, and the plant. For example, as shown in the Examples below, it is possible to provide ripe red fruits containing GABA at a high concentration of about 150 mg / 100 g FW (about 13 times that of the wild type), and tomatoes that bear these fruits.

[0021] This figure shows an outline of a cassette for expressing four types of guide RNAs (gRNA1 to gRNA4) for genome editing of a regulatory sequence located upstream of the tomato γ-aminobutyric acid aminotransferase 1 gene (SlGABA-T1 gene) and Cas9. ０ This figure shows an overview of a scheme for obtaining null segregants homozygously carrying mutations introduced into the regulatory sequence from tomato individuals. This figure shows an overview of the locations of gRNA1 to gRNA4 in the regulatory sequence of the SlGABA-T1 gene and the introduced deletion mutation. This graph shows the results of qRT-PCR analysis of the expression level of the SlGABA-T1 gene in fruit (immature stage, breaker stage, red ripe stage) for wild-type tomato, genome-edited tomato (SlGAD3ΔC) in which the C-terminal autoinhibitory domain in GAD3 has been deleted, and a tomato line (SlGAD3ΔC + SlGABA-T1Δpro) in which the deletion mutation has been introduced into the regulatory sequence of the GABA-T1 gene against the SlGAD3ΔC background. In the figure, the vertical axis shows the relative expression value of the SlGABA-T1 gene to the housekeeping gene (ubiquitin gene). 1 is a graph showing the results of measuring the GABA content in fruits (ripe red stage) of wild-type tomato, SlGAD3ΔC, and SlGAD3ΔC+SlGABA-T1Δpro. In the graph, the vertical axis represents the GABA content per 100 g of fresh fruit. FIG. 1 is a graph showing the results of measuring the plant height of wild-type tomato, SlGAD3ΔC, and SlGAD3ΔC+SlGABA-T1Δpro. In the graph, the vertical axis represents the length from the cotyledon to the meristem, measured while the plant was held upright 80 days after sowing.

[0022] As will be shown in the Examples below, it has been discovered that the GABA content in plants can be increased by introducing a mutation into a regulatory sequence upstream of the GABA-T gene and suppressing the expression level of the gene. On the other hand, it has been revealed that introducing a mutation into the regulatory sequence does not result in sterility or dwarfism, leading to the completion of the present invention. Therefore, the present invention relates to a method for producing a plant with an improved GABA content, comprising: (1) a step of introducing a mutation into a regulatory sequence located upstream of the GABA-T gene in a plant cell to suppress expression of the gene; and (2) a step of regenerating a plant from the plant cell in which expression of the gene has been suppressed in step (1).

[0023] The "plants" that are the subject of the present invention are not particularly limited as long as they are capable of biosynthesizing GABA, and include, for example, seed plants including angiosperms and gymnosperms. Angiosperms include dicotyledonous and monocotyledonous plants. "Dicocotyledonous plants" include, for example, Solanaceae, Cucurbitaceae, Leguminosae, Cruciferae, Convolvulaceae, Rosaceae, Asteraceae, Amaranthaceae, Polygonaceae, Moraceae, and Malvaceae. "Solanaceae plants" include tomato, potato, eggplant, tobacco, chili pepper, etc. "Cucurbitaceae plants" include melon, pumpkin, watermelon, Japanese cantaloupe, cucumber, etc. "Legumes" include soybean, adzuki bean, kidney bean, pea, cowpea, alfalfa, etc. "Brassicaceae plants" include cabbage, cauliflower, radish, Chinese cabbage, rapeseed, etc. Examples of "Convolvulaceae plants" include sweet potato (sweet potato). Examples of "Rosaceae plants" include strawberries and apples. Examples of "Asteraceae plants" include lettuce. Examples of "Amaranthaceae plants" include sugar beets (sugar beets). Examples of "Polygonaceae plants" include buckwheat. Examples of "Moraceae plants" include mulberry, fig, and rubber tree. Examples of "Mallow family plants" include cotton and kenaf. Examples of "monocotyledonous plants" include any species, such as grasses, lilies, Musaceae, Bromeliaceae, and orchids. Examples of "Gramineae plants" include rice, wheat, barley, corn, oats, turfgrass, sorghum, rye, foxtail millet, and sugarcane. Examples of "Liliaceae plants" include leeks and asparagus. Examples of "Musaceae plants" include bananas. Examples of "Bromeliaceae plants" include pineapples, etc. Examples of "Orchidaceae plants" include orchids, etc. Examples of "gymnosperms" include ginkgo, pine, cedar, cycad, etc. As plants according to the present invention, tomato, melon, rice, potato, soybean, and pumpkin are preferred, with tomato being more preferred, from the viewpoint that GABA tends to be easily contained in the edible parts.

[0024] The plants of the present invention may be wild species or cultivated species. Furthermore, they may be genetically modified or genome-edited versions of these plants (e.g., disease-resistant crops, herbicide-resistant crops, pest-resistant crops, crops with improved taste, crops with improved shelf life, or crops with improved yield). Thus, as shown in the Examples below, examples of plants that are the subject of the present invention include plants in which the function of the C-terminal autoinhibitory domain in GAD described below has been lost.

[0025] In the present invention, "GABA," the target of content enhancement, is a type of amino acid, also known as γ-aminobutyric acid or 4-aminobutyric acid. "Improvement of GABA concentration" refers to, for example, an improvement in GABA concentration compared to a plant before the introduction of a mutation, as described below. Preferably, the GABA concentration is improved in the edible portion of the plant compared to that of the plant before the introduction of a mutation, as described below. "Improvement" refers to an increase in GABA concentration, preferably 1.2 times or more (e.g., 1.5 times or more, 1.7 times or more, 2 times or more, 3 times or more, 4 times or more), more preferably 5 times or more (e.g., 6 times or more, 7 times or more, 8 times or more, 9 times or more), and even more preferably 10 times or more (e.g., 11 times or more, 12 times or more, 13 times or more, 14 times or more, 15 times or more), compared to the plant or its edible portion before the introduction of a mutation. "Edible parts" refers to the parts of a plant that can be eaten, and varies depending on the type of plant. Examples include fruits, seeds, stems (tubers, etc.), roots (tuberous roots, etc.), leaves, buds (for example, young shoots (sprouts, etc.)), and flowers. In the present invention, a plant with an improved GABA concentration can also be referred to as a plant that contains a high concentration of GABA. Here, "high concentration" refers to, for example, the GABA content in tomato fruit, preferably 100 mg / 100 g FW or more, more preferably 110 mg / 100 g FW or more, even more preferably 120 mg / 100 g FW or more, more preferably 130 mg / 100 g FW or more, even more preferably 140 mg / 100 g FW or more, and more preferably 150 mg / 100 g FW or more. The unit "mg / 100 g FW" here represents the GABA content per 100 g of fresh fruit at the red fruit stage.

[0026] In the present invention, the target of expression inhibition, "γ-aminobutyrate aminotransferase (GABA-T)," is an enzyme (registered as EC number 2.6.1.96) that catalyzes the conversion of GABA to succinic semialdehyde (SSA). Examples include proteins belonging to the "Gamma aminobutyrate transaminase" group on UniProt, and proteins encoded by genes belonging to the "GABA-TP" group. A specific example, in the case of tomato, is the protein identified by UniProt accession Q84P54. Table 1 shows GABA-T genes derived from various plants, along with their respective registration IDs (gene IDs) in reference databases.

[0027]

[0028] "Suppression of GABA-T gene expression" means, for example, that the expression level of the GABA-T gene is reduced compared to the plant or edible parts thereof before the introduction of the mutation described below. Here, "expression level" may be the amount of mRNA (or cDNA), or may be the amount of protein reflecting this. "Reduction" means that the expression level of the GABA-T gene is preferably 90% or less (e.g., 80% or less, 70% or less, 60% or less), more preferably 50% or less (e.g., 40% or less, 30% or less), and even more preferably 20% or less (e.g., 15% or less, 10% or less), compared to the plant or edible parts thereof before the introduction of the mutation.

[0029] In the present invention, the "regulatory sequence" located upstream of the GABA-T gene into which the mutation described below is introduced refers to a sequence located 5' from the start codon in the DNA sequence encoding the GABA-T protein in the genomic sequence (preferably a sequence 5' from the transcription initiation site), and is a DNA sequence capable of regulating the expression (transcription, etc.) of the gene. Examples of regulatory sequences located upstream of the GABA-T gene in various plants include the DNA sequences specified by the SEQ ID NOs shown in Table 1. Furthermore, with regard to tomato, as shown in the Examples below, an example is the DNA sequence consisting of positions 2056 to 2422 of SEQ ID NO: 1, and more preferably the DNA sequence consisting of positions 2056 to 2314 of SEQ ID NO: 1.

[0030] Furthermore, the DNA sequence consisting of positions 2056 to 2422 of SEQ ID NO: 1, as shown in the Examples below, contains a TATA BOX and an initiator as sequences capable of regulating the amount of transcription. Specifically, the TATA BOXes include TATABOX2 (PLACE ID: S000109, DNA sequence: TATAAAT), TATABOX3 (PLACE ID: S000110, DNA sequence: TATTAAT), and TATABOX5 (PLACE ID: S000203, DNA sequence: TTATTT). The initiator includes INRNTPSADB (PLACE ID: S000395, DNA sequence: YTCANTYY). Meanwhile, all of the DNA sequences specified by the above SEQ ID NOs: 16 to 30 also contain such a TATA BOX and initiator. Therefore, these sequences can be suitable targets for mutagenesis.

[0031] Note that DNA sequences can vary in nature. Furthermore, sequences related to the same gene of the same species can differ (genetic polymorphisms). Therefore, in the present invention, the regulatory sequences into which the mutations described below are introduced are not limited to the above-mentioned typical DNA sequences, but can also include sequences corresponding to them. Here, "corresponding sequences" refers to DNA sequences that are identical to the above-mentioned typical DNA sequences (e.g., DNA sequences specified by the above-mentioned SEQ ID NOs or partial sequences thereof) or have high homology or identity thereto. Here, "high" refers to at least 80% or more, preferably 85% or more, more preferably 90% or more (e.g., 91% or more, 92% or more, 93% or more, 94% or more), and even more preferably 95% or more (e.g., 96% or more, 97% or more, 98% or more, 99% or more). Sequence homology or identity can be determined, for example, using the BLAST program (Altschul et al. J. Mol. Biol., 215:403-410, 1990). The program is based on the BLAST algorithm by Karlin and Altschul (Proc. Natl. Acad. Sci. USA, 87:2264-2268, 1990, Proc. Natl. Acad. Sci. USA, 90:5873-5877, 1993). Specific techniques for such analysis are known, but when analyzing a DNA sequence using BLAST, the parameters are, for example, score = 100 and wordlength = 12. Furthermore, when implementing the BLAST program, the default parameters of the program can also be used.

[0032] In the present invention, examples of mutations introduced into the above-mentioned regulatory sequence include deletion, insertion, and / or substitution of one or more DNA fragments, preferably deletion of one or more DNA fragments. Here, the term "multiple" is not particularly limited as long as it can suppress the expression (transcription, etc.) of the GABA-T gene and improve the GABA content. For example, the lower limit can be 2 or more bases, 3 or more bases, 5 or more bases, 10 or more bases, 20 or more bases, 30 or more bases, 50 or more bases, 100 or more bases, 200 or more bases, 500 or more bases, 1000 or more bases, or 2000 or more bases. The upper limit can be, for example, 2500 or less bases, 2000 or less bases, 1500 or less bases, 1000 or less bases, 500 or less bases, 200 or less bases, 100 or less bases, 50 or less bases, 40 or less bases, 20 or less bases, 10 or less bases, or 5 or less bases. More specific examples of the number of bases for deletion, insertion, and / or substitution introduced into the above-mentioned regulatory sequence include 2 to 2500 bases, 20 to 2000 bases, 50 to 1500 bases, 100 to 1000 bases, and 200 to 500 bases (250 to 450 bases, 300 to 400 bases, and 350 to 370 bases (particularly 367 bases)). Further examples include 2 to 300 bases, 3 to 200 bases, 5 to 100 bases, 10 to 50 bases, and 30 to 40 bases. Furthermore, the number of mutations (number of positions) introduced into the regulatory sequence in the present invention is not particularly limited, as long as it is possible to suppress the expression (transcription, etc.) of the GABA-T gene and improve the GABA content, and may be one or multiple (for example, 2, 3 or less, 5 or less, 10 or less, 20 or less, 30 or less, 40 or less, 50 or less).

[0033] Those skilled in the art can introduce mutations into the regulatory sequences of the present invention using known mutagenesis methods, including, but not limited to, genome editing, physical mutagenesis, methods using chemical mutagens, and methods for introducing transposons or the like into genomic DNA.

[0034] "Genome editing" is a method of modifying a target gene using a site-specific nuclease (e.g., a DNA double-strand cleavage enzyme such as zinc finger nuclease (ZFN), transcription activation-like effector nuclease (TALEN), or CRISPR-Cas enzyme). For example, fusion proteins such as ZFNs (U.S. Patent Nos. 6,265,196, 8,524,500, 7,888,121, and European Patent No. 1,720,995), TALENs (U.S. Patent Nos. 8,470,973 and 8,586,363), and nuclease domain-fused pentatricopeptiderepeat (PPR) (Nakamura et al., Plant Cell Physiol 53:1171-1179 (2012)), CRISPR-Cas9 (U.S. Patent No. 8,697,359, and International Publication No. 2013 / 176772), CRISPR-Cpf1 (Zetsche B. et al., al., Cell, 163(3):759-71, (2015)) and Target-AID (K. Nishida et al., Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems, Science, DOI: 10.1126 / science.aaf8729, (2016)) or a method using a guide RNA and protein complex, or a protein complex.

[0035] The "Cas enzyme" is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include type I CRISPR enzymes, type II CRISPR enzymes, and type III CRISPR enzymes, with Cas9, which is a type II CRISPR enzyme, being preferred. The "Cas9" is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include Cas9 from Streptococcus pneumoniae, Cas9 from Streptococcus pyogenes, and Cas9 from S. Examples of Cas9 include Streptococcus thermophilus Cas9 and Staphylococcus aureus Cas9, but Streptococcus pyogenes Cas9 (SpCas9) is preferred. Furthermore, it may be a mutant of Cas9 derived from these organisms, or it may be a D10A mutant of Cas9, which is known to function as a nickase (a DNA cleavage enzyme that nicks only one DNA strand), or it may be a Cas9 homolog or ortholog.

[0036] Examples of "physical mutagenesis methods" include heavy ion beam (HIB) irradiation, fast neutron irradiation, gamma ray irradiation, and ultraviolet irradiation (see Hayashi et al., Cyclotrons and Their Applications, 2007, 18th International Conference, pp. 237-239, and Kazama et al., Plant Biotechnology, 2008, Vol. 25, pp. 113-117).

[0037] An example of a "method using a chemical mutagen" is a method of treating seeds, etc. with a chemical mutagen (see, for example, Zwar and Chandler, Planta, 1995, vol. 197, pp. 39-48). Chemical mutagens are not particularly limited, and examples include N-methyl-N-nitrosourea (MNU), ethyl methanesulfonate (EMS), N-ethyl-N-nitrosourea (ENU), sodium azide, sodium bisulfite, hydroxylamine, N-methyl-N'-nitro-N-nitroguanidine (MNNG), N-methyl-N'-nitrosoguanidine (NTG), O-methylhydroxylamine, nitrous acid, formic acid, and nucleotide analogs.

[0038] Examples of "methods for introducing transposons into genomic DNA" include, for example, T ＯＳ Examples of such methods include inserting transposons such as 17, T-DNA, etc. into the genomic DNA of plants (see Kumar et al., Trends Plant Sci., 2001, Vol. 6, No. 3, pp. 127-134, and Tamara et al., Trends in Plant Science, 1999, Vol. 4, No. 3, pp. 90-96).

[0039] For plants into which mutations have been introduced by the above methods, it is possible to confirm that a mutation has been introduced into the regulatory sequence of the present invention by known methods. Examples of such known methods include DNA sequencing (next-generation sequencing, etc.), PCR, microarray analysis, and Southern blotting. Using such methods, it is possible to determine whether a mutation has been introduced into the regulatory sequence of the present invention by comparing the regulatory sequence or its length before and after the introduction of the mutation.

[0040] Another method for confirming that a mutation has been introduced into the regulatory sequence of the present invention is TILLING (Targeting Induced Local Lesions IN Genomes) (see Slade et al., Transgenic Res., 2005, Vol. 14, pp. 109-115, and Comai et al., Plant J., 2004, Vol. 37, pp. 778-786). In particular, when a non-selective mutation has been introduced into the genome of a plant using the aforementioned heavy ion beam irradiation or chemical mutagen, the regulatory sequence of the present invention or a part thereof can be amplified by PCR, and then individuals having a mutation in the amplification product can be selected by the TILLING method or the like.

[0041] Furthermore, by crossing a plant into which a mutation has been introduced by the above-mentioned method with a wild-type plant and performing backcrossing, it is also possible to remove mutations introduced into regions other than the target regulatory sequence of the present invention.

[0042] A plant having an improved GABA concentration due to the introduction of a mutation into the regulatory sequence of the present invention may be a heterozygote consisting of a wild-type plant and a mutation in the regulatory sequence of the present invention. In such cases, for example, such heterozygotes are crossed to obtain F1 plants, and homozygotes having the regulatory sequence of the GABA-T gene into which the mutation has been introduced are selected from the F1 plants. In this case, the term "homozygote plant having the regulatory sequence of the GABA-T gene into which the mutation has been introduced" includes not only plants having two regulatory sequences (alleles) of the GABA-T gene having identical mutations, but also plants having a regulatory sequence of a first GABA-T gene having a first mutation and a regulatory sequence of a second GABA-T gene having a second mutation.

[0043] In the present invention, introduction of a mutation into a regulatory sequence according to the present invention can be carried out in a plant, a seed, or a plant cell according to the above-mentioned method. Plant cells include not only cultured cells derived from plants but also cells within the plant. Furthermore, plant cells in various forms, such as suspension culture cells, protoplasts, leaf segments, callus, immature embryos, and pollen, are also included.

[0044] Furthermore, in the present invention, the above-mentioned DNA encoding the site-specific nuclease, fusion protein, or guide RNA-protein complex, DNA encoding a transposon, etc. may be introduced into plant cells in the form of being inserted into a vector.

[0045] The vector into which the DNA for introducing a mutation into the regulatory sequence of the present invention is inserted is not particularly limited as long as it is capable of expressing the inserted gene in plant cells, and may contain a promoter for constitutive or inducible expression of the DNA. Examples of promoters for constitutive expression include the rice ubiquitin promoter, the cauliflower mosaic virus 35S promoter, the rice actin promoter, and the maize ubiquitin promoter. Examples of promoters for inducible expression include promoters known to be expressed in response to external factors such as infection or invasion by filamentous fungi, bacteria, or viruses, low temperature, high temperature, drought, ultraviolet radiation, and spraying with specific compounds. Furthermore, pol III promoters such as the U6 promoter are preferably used as promoters for expressing DNA encoding short RNAs such as guide RNAs as DNA of the present invention.

[0046] As a method for introducing the DNA or a vector into which the DNA has been inserted into a plant cell, various methods known to those skilled in the art can be used, such as an Agrobacterium-mediated method (Agrobacterium method), particle bombardment, polyethylene glycol method, electroporation, etc. Mutations can also be introduced into plant cells without taking the form of DNA, by introducing the above-mentioned site-specific nucleases, fusion proteins, and transposons as proteins, or the above-mentioned guide RNAs as RNAs.

[0047] Thus, in the present invention, the GABA content in plants can be increased by using a substance that targets the regulatory sequence of the present invention, such as the DNA, a vector into which the DNA has been inserted, the protein, or the RNA. Therefore, the present invention can also provide a drug for increasing the GABA content in plants, comprising as an active ingredient at least one substance that targets the regulatory sequence of the GABA-T gene of the present invention, selected from the group consisting of the DNA, a vector into which the DNA has been inserted, the protein, and the RNA.

[0048] Such a pharmaceutical preparation may be in an embodiment in which the two active ingredients are contained in a single composition, or in an embodiment in which the two active ingredients are contained in separate compositions (a so-called kit). In addition to the above substances, the pharmaceutical preparation of the present invention may also contain other ingredients such as a buffer solution, a stabilizer, a preservative, an antiseptic, etc.

[0049] Furthermore, plants with improved GABA content can be obtained by regenerating plants from cells into which a mutation has been introduced into the regulatory sequence of the present invention using the above-mentioned methods. For example, in the case of tomato, methods such as those described in Sun et al., Plant Cell Physiology, 2006; 47(3):426-431 and Sonia Hamza and Yves Chupeau, J. Exp. Bot., 44:1837-1845, 1993 can be used. Furthermore, even for other plant species, transformation and plant regeneration can be performed using the method described in Tabei et al. (Yutaka Tabei, ed., "Transformation Protocols [Plant Edition]," Kagaku Dojin Co., Ltd., published September 20, 2012).

[0050] Furthermore, edible parts having an improved GABA concentration can be obtained from the plant obtained in this manner. Therefore, the present invention also provides a method for producing edible parts having an improved GABA concentration, which includes the steps of producing a plant having an improved GABA concentration by the above-mentioned method and obtaining edible parts from the plant. The edible parts are as described above, and those skilled in the art can obtain them from plants by appropriately preparing them depending on the type of plant or edible parts.

[0051] Furthermore, by using the above-mentioned method, etc., it is possible to obtain a plant having an improved GABA concentration by artificially introducing a mutation into the regulatory sequence of the present invention. Thus, the present invention relates to a plant or edible part thereof having an improved GABA concentration, in which a mutation has been artificially introduced into the regulatory sequence located upstream of the GABA-T gene, thereby suppressing expression of the gene.

[0052] Such plants do not suffer from sterility or dwarfing, as reported in the degradation of GABA-T mRNA by RNAi (Non-Patent Document 4). Here, "sterility" means that seeds capable of developing into plants of the next generation are not produced. Furthermore, "dwarfing" means that the plant matures while remaining small compared to the plant before the introduction of the mutation in the present invention, and "no dwarfing" may also include a state in which the degree of dwarfing is small. More specifically, for example, in tomato, the plant height after the flowering stage (approximately 40 days after sowing, for example, 80 days after sowing), when the plant switches from vegetative growth to reproductive growth, is 70% or less compared to the plant before the introduction of the mutation in the present invention.

[0053] The regulatory sequence of the present invention, the introduction of mutations therein, and the plant or edible portion thereof having an improved GABA concentration due to the introduction of the mutations are as described above. Once a plant having an improved GABA concentration is obtained by artificially introducing mutations into the regulatory sequence of the present invention, it is possible to obtain progeny from the plant through sexual or asexual reproduction. Furthermore, propagation materials (e.g., seeds, cuttings, stems, calli, protoplasts, etc.) can be obtained from the plant, its progeny, or clones, and used to mass-produce the plant. Therefore, the present invention includes the progeny and clones of plants having an improved GABA concentration, as well as their propagation materials. Examples of propagation materials include seeds, stems, calli, and protoplasts.

[0054] Furthermore, in the sexual reproduction, by crossing with other lines (other varieties, etc.), it is possible to further improve the GABA concentration and to obtain progeny that are endowed with other traits (e.g., disease resistance, herbicide resistance, pest resistance, improved taste, improved storability, improved yield). From the viewpoint of further improving the GABA concentration, for example, as shown in the Examples below, crossing with a plant in which the function of the C-terminal autoinhibitory domain in GAD has been lost can be mentioned. By this crossing, a mutation is artificially introduced into the regulatory sequence located upstream of the GABA-T gene, thereby suppressing the expression of the gene and causing the function of the C-terminal autoinhibitory domain in GAD to be lost, thereby making it possible to obtain a plant with an improved GABA concentration.

[0055] In the present invention, "glutamate decarboxylase (GAD)" refers to an enzyme (registered as EC number 4.1.1.15) that catalyzes the conversion of glutamic acid to GABA. Examples include proteins belonging to the "Glutamate decarboxylase" category on UniProt, and proteins encoded by genes belonging to the "GAD" category. A specific example is a protein derived from tomato identified by UniProt accession number B1Q3F2. Table 2 lists GAD genes derived from various plants, along with their respective registration IDs (gene IDs) in reference databases. The "C-terminal autoinhibitory domain" targeted for loss of function in GAD is a calmodulin-binding domain (CaM BD) consisting of approximately 30 amino acids (e.g., 22-25 amino acids) located at the C-terminus, which functions as an autoinhibitory (autoinhibitory) domain for inactivating the enzymatic activity of GAD. Such loss of function can be exemplified by the deletion of all or part of the C-terminal autoinhibitory domain. Furthermore, those skilled in the art can appropriately prepare such loss-of-function plants or plant cells using known techniques such as genome editing, as well as the introduction of mutations targeting the regulatory sequence of the GABA-T gene (see, for example, Non-Patent Document 3).

[0056]

[0057] The present invention will be described in more detail below based on examples, but the present invention is not limited to the following examples. The sequences of the DNA encoding the gRNA and the primers used in each experiment shown below are shown in Table 3.

[0058]

[0059] Genome editing targeting the upstream sequence of a GABA metabolism-related gene was used as the test plant: Micro-Tom, a dwarf model variety of tomato (Solanum lycopersicum L.). Using Sol Genomics Network (https: / / solgenomics.net), the genomic sequence of 2,500 bases upstream of the start codon of a GABA metabolism-related gene (tomato-derived γ-aminobutyric acid aminotransferase (GABA-T) gene (SlGABA-T1 (Solyc07g043310) gene)) was obtained (SEQ ID NO: 1).

[0060] Four gRNAs were designed using PAM sequences (NGG) located 81 bases, 448 bases, 1,103 bases, and 1,382 bases upstream from the start codon (SEQ ID NOs: 2, 3, 4, and 5), and vectors containing these four target sequences in the expression cassette were constructed (Figure 1). Transformation was performed using the Agrobacterium method optimized for Micro-Tom (Sun et al., Plant cell psychiatry, 2006; 47(3):426-431). The Agrobacterium vector was then introduced into a cotyledon explant of Micro-Tom (wild-type), and the expression cassette shown in Figure 1 was introduced into its nuclear genome. Plants regenerated from a series of selective media containing kanamycin were initially selected as candidates for transformants.

[0061] Next, to confirm that the transformants were present, genomic DNA was extracted from young tomato leaves, and using primers (SEQ ID NOs: 6 and 7) that specifically bind to the CRISPR / Cas9 vector, it was confirmed whether the CRISPR / Cas9 vector had been introduced into the tomato nuclear genome.

[0062] Sanger sequencing was also performed to confirm whether mutations occurred near the target sequence. First, DNA fragments near the target sequence were PCR-amplified using genomic DNA extracted from tomato and primers designed around the target sequence (SEQ ID NOs: 8, 9, 10, and 11). The amplified PCR fragments were used as sequencing templates and subjected to Sanger sequencing using primers specific to the SlGABA-T1 upstream sequence (SEQ ID NO: 8 was used for analysis of gRNA1, SEQ ID NO: 9 for analysis of gRNA2, SEQ ID NO: 10 for analysis of gRNA3, and SEQ ID NO: 11 for analysis of gRNA4).

[0063] As a result, introduction of the CRISPR / Cas9 vector and introduction of a mutation were confirmed in one line.

[0064] <Analysis of genome-edited GABA metabolism-related genes> Next, to increase the variation of mutant alleles, one TO individual line containing the CRISPR / Cas9 vector was crossed with a Micro-Tom SlGAD3 mutant in which GABA accumulation in fruit was improved by genome editing of wild-type Micro-Tom or SlGAD3. The F1 line obtained by crossing was further selfed, and the progeny were homozygous for the mutation and null segregants in which the CRISPR / Cas9 vector had been removed were obtained (Figure 2).

[0065] In the SlGAD3 mutant, the C-terminal autoinhibitory domain of GAD3 is deleted by genome editing. See Nonaka et al., Scientific Reports, 2017; 7(1), 7057. Hereinafter, the SlGAD3 mutant will also be referred to as "SlGAD3ΔC"). The amino acid sequences of the wild-type and SlGAD3ΔC are shown in SEQ ID NOs: 14 and 15, respectively. The C-terminal autoinhibitory domain is the 30 amino acids from asparagine at position 455 to the last amino acid (cysteine) at position 484 in the amino acid sequence described in SEQ ID NO: 14.

[0066] Subsequently, the obtained mutant homozygous nulligrant individuals were cultivated in a closed cultivation room, and the GABA content of the fruit was measured and the expression of the SlGABA-T1 gene was analyzed. A GABA measurement kit (Enzyme Sensor Co., Ltd.) was used to measure the GABA content. The method followed the manual that came with the kit. In addition, qRT-PCR was used to analyze the expression of the SlGABA-T1 gene, and SlGABA-T1-specific primers (SEQ ID NOs: 12 and 13) were used as primers.

[0067] Then, in the background of SlGAD3ΔC, a line in which the region between gRNA1 and gRNA2 (the region consisting of 79 to 445 bases from the start codon (the region consisting of 2056 to 2422 bases in the base sequence described in SEQ ID NO: 1): 367 bases) was deleted (Figure 3, hereinafter also referred to as "SlGAD3ΔC + SlGABA-T1Δpro") was analyzed. As a result, suppression of expression of the SlGABA-T1 gene in fruit was observed (Figure 4, compared to the wild type, 79% at the immature stage, 66% at the color break stage, and 33% at the red ripe stage). Furthermore, an increase in the amount of GABA accumulated in fruit was observed (Figure 5, 13-fold increase compared to the wild type and 2-fold increase compared to SlGAD3ΔC at the red ripe stage). On the other hand, dwarfism and sterility traits were not confirmed in SlGAD3ΔC + SlGABA-T1Δpro (Figure 6).

[0068] As described above, according to the present invention, by introducing a mutation into the regulatory sequence upstream of the GABA-T gene, it is possible to increase the GABA content without causing any adverse traits. Therefore, the present invention is useful for suppressing elevated blood pressure, alleviating stress, improving sleep quality, maintaining skin elasticity, etc.

Claims

1. A method for producing a plant with an improved concentration of gamma-aminobutyric acid (GABA), comprising: (1) a step of introducing a mutation into a regulatory sequence located upstream of the gamma-aminobutyric acid aminotransferase (GABA-T) gene in a plant cell to suppress expression of the gene; and (2) a step of regenerating a plant from the plant cell in which expression of the gene has been suppressed in step (1).

2. The method of claim 1, wherein the plant cells are tomato cells and the mutation is introduced into the DNA sequence set forth in SEQ ID NO: 1 or a sequence corresponding thereto in the regulatory sequence.

3. The method of claim 1, wherein the plant cells are tomato cells, and the mutation is introduced into a DNA sequence consisting of positions 2056 to 2422 of SEQ ID NO: 1 in the regulatory sequence, or a sequence corresponding thereto.

4. (3) The production method described in claim 1, further comprising the step of crossing the plant obtained in step (2) with a plant in which the function of the C-terminal autoinhibitory domain in glutamic acid decarboxylase (GAD) has been lost.

5. A method for producing edible parts with an improved GABA content, comprising the steps of producing a plant body with an improved GABA content by the method described in any one of claims 1 to 4, and obtaining edible parts from the plant body.

6. A plant with an improved GABA concentration, in which a mutation has been artificially introduced into the regulatory sequence located upstream of the GABA-T gene, thereby suppressing the expression of said gene.

7. The plant according to claim 6, which is a tomato plant, in which the mutation has been introduced into the DNA sequence set forth in SEQ ID NO: 1 or a sequence corresponding thereto in the regulatory sequence.

8. The plant according to claim 6, which is a tomato plant, in which the mutation has been introduced into a DNA sequence consisting of positions 2056 to 2422 of SEQ ID NO: 1 in the regulatory sequence or a sequence corresponding thereto.

9. The plant body according to claim 6, further comprising a loss of function of the C-terminal autoinhibitory domain in GAD.

10. An edible portion of a plant body according to any one of claims 6 to 9.

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