Plant having increased γ-aminobutyric acid content and method for producing the same

Abstract

To provide a method for producing a plant with increased γ-aminobutyric acid content without causing sterility or dwarfism, and to provide the plant.SOLUTION: It has been found that, in a plant, introducing a mutation into a regulatory sequence upstream of a GABA-T gene and suppressing an expression level of the gene enable an increase in γ-aminobutyric acid content without causing sterility or dwarfism.SELECTED DRAWING: None

Description

[Technical Field]

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

[0002] GABA is an amino acid that functions as a neurotransmitter in the human central nervous system, but when taken orally, it is also known to be effective in suppressing high blood pressure, relieving stress, improving sleep quality, maintaining skin elasticity, etc. For this reason, 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 GABA accumulation in plants.

[0003] For example, enhancing GABA biosynthesis or inhibiting GABA degradation is thought to be effective methods for increasing GABA accumulation in tomato fruits. Reported methods for enhancing GABA biosynthesis include overexpressing the glutamic acid decarboxylase (GAD) gene, which is 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 RNA interference (RNAi) is used to suppress the expression of the GABA-degrading enzyme (γ-aminobutyric acid aminotransferase: GABA-T) (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. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Takayama et al. 2015 Plant Cell Physiol 56,1533-1545 [Non-patent document 2] Takayama et al. 2017 Plant Cell Rep 36,103-116 [Non-patent document 3] Nonaka et al. 2017 Sci Rep 7.1, 7057 [Non-patent document 4] Koike et al. 2013 Plant Cell Physiol 54,793-807 Summary of the Invention [Problem to be solved by the invention]

[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. [Means for solving the problem]

[0007] As a result of extensive research aimed at achieving 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. On the other hand, they have shown that the sterility and dwarfism reported in RNAi-mediated degradation of GABA-T mRNA (Non-Patent Document 4) do not occur when the 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) 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; (2) a step of regenerating a plant from the plant cells in which the expression of the gene has been suppressed in step (1).

[0010] [2] The method of producing according to [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.

[0011] [3] The method of producing according to [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.

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

[0013] [5] (3) A manufacturing method described in any one of [1] to [3], further comprising a step of crossing the plant obtained in 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 described in any one of [1] to [5], and obtaining an edible part from the plant body.

[0015] [7] 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, suppressing the expression of the gene.

[0016] [8] The plant according to [7], 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.

[0017] [9] The plant according to [7], 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.

[0018]

[10] A plant body 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 described in any one of [7] to

[10] . [Effects of the Invention]

[0020] According to the present invention, it is possible to provide a method for producing a plant with an improved GABA concentration without causing sterility or dwarfing, 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 gFW (about 13 times that of the wild type), and tomatoes that bear these fruits. [Brief explanation of the drawings]

[0021] [Figure 1] This diagram shows an overview of the cassettes for expressing four types of guide RNAs (gRNA1 to gRNA4) and Cas9 for genome editing of the regulatory sequence located upstream of the tomato γ-aminobutyric acid aminotransferase 1 gene (SlGABA-T1 gene). [Figure 2] FIG. 2 shows an outline of a scheme for obtaining null segregants homozygous for the mutations introduced into the regulatory sequences from the TO tomato individuals carrying the CRISPR / Cas9 expression cassette shown in FIG. 1. [Figure 3] FIG. 1 is a diagram showing an overview of the locations of gRNA1 to gRNA4 in the regulatory sequence of the SlGABA-T1 gene and the introduced deletion mutations. [Figure 4]This graph shows the results of qRT-PCR analysis of SlGABA-T1 gene expression in fruit (immature, breakthrough, and red-ripening stages) for wild-type tomatoes, genome-edited tomatoes lacking the C-terminal autoinhibitory domain of GAD3 (SlGAD3ΔC), and a tomato line with the SlGAD3ΔC background and the deletion mutation introduced into the GABA-T1 regulatory sequence (SlGAD3ΔC+SlGABA-T1Δpro). The vertical axis indicates the relative expression level of SlGABA-T1 gene to that of a housekeeping gene (ubiquitin gene). [Figure 5] 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, where the vertical axis represents the GABA content per 100 g of fresh fruit. [Figure 6] 1 is a graph showing the results of measuring the plant height of wild-type tomato, SlGAD3ΔC, and SlGAD3ΔC+SlGABA-T1Δpro, where the vertical axis represents the length from the cotyledon to the meristem, measured while the plant was held upright 80 days after sowing. DETAILED DESCRIPTION OF THE INVENTION

[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 the 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 sterility and dwarfism do not occur when a mutation is introduced into the regulatory sequence, leading to the completion of the present invention. Therefore, the present invention provides a method for producing a plant with an improved GABA concentration, which comprises: (1) introducing a mutation into a regulatory sequence located upstream of a GABA-T gene in a plant cell to suppress expression of the gene; (2) a step of regenerating a plant from the plant cells in which the expression of the gene has been suppressed in the step (1).

[0023] The "plants" 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. "Cruciferous 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 and the like. Examples of "Amaranthaceae plants" include sugar beets (sugar beets). Examples of "Polygonaceae plants" include buckwheat and the like. 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, but examples include 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 and the like. "Bromeliaceae plants" include pineapples, etc. "Orchidaceae plants" include orchids, etc. "Gymnosperms" include ginkgo, pine, cedar, cycad, etc. As plants according to the present invention, tomatoes, melons, rice, potatoes, soybeans, and pumpkins are preferred, from the viewpoint that GABA tends to be easily contained in the edible parts. Tomatoes are more preferred.

[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, 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, as described below, has been lost, as shown in the Examples below.

[0025] In the present invention, "GABA," the target of the increased content, is a type of amino acid, also known as γ-aminobutyric acid or 4-aminobutyric acid. "Improved GABA concentration" refers to, for example, an increased GABA concentration compared to a plant before the introduction of a mutation, as described below. Preferably, the GABA concentration in the edible portion of the plant is increased compared to that in the plant before the introduction of a mutation, as described below. "Improved" refers to an increase in GABA concentration of 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 before the introduction of a mutation or its edible portion. "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 (e.g., 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 number Q84P54. Furthermore, Table 1 shows the reference databases for GABA-T genes derived from various plants, along with their respective registration IDs (gene IDs).

[0027] [Table 1]

[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, as described below. Here, "expression level" may refer to the amount of mRNA (or cDNA), or may also refer to the amount of protein that reflects 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 identified by the SEQ ID NOs: listed 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 SEQ ID NOs: 16 to 30 above also contain such a TATA BOX and initiator. Therefore, these sequences can be suitable targets for mutagenesis.

[0031] In nature, DNA sequences can vary. Furthermore, even sequences related to the same gene of the same species can vary (genetic polymorphism). 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 word length = 12. The default parameters of the BLAST program can also be used when running the program.

[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 may 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 may 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 specifically, the number of bases for deletion, insertion, and / or substitution introduced into the above-mentioned regulatory sequence may be, for example, 2 to 2500 bases, 20 to 2000 bases, 50 to 1500 bases, 100 to 1000 bases, or 200 to 500 bases (250 to 450 bases, 300 to 400 bases, or 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 sites) 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 increase the GABA content, and may be one or more (for example, 2, 3 or less, 5 or less, 10 or less, 20 or less, 30 or less, 40 or less, or 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 target genes using site-specific nucleases (e.g., zinc finger nucleases (ZFNs), transcription activation-like effector nucleases (TALENs), CRISPR-Cas enzymes, and other DNA double-strand break enzymes). For example, fusion proteins such as ZFNs (U.S. Patent Nos. 6,265,196, 8,524,500, 7,888,121, European Patent No. 1,720,995), TALENs (U.S. Patent Nos. 8,470,973, 8,586,363), and nuclease domain-fused PPR (pentatricopeptide repeat) (Nakamura et al., Plant Cell Physiol 53:1171-1179 (2012)), CRISPR-Cas9 (U.S. Patent No. 8,697,359, International Publication No. 2013 / 176772), CRISPR-Cpf1 (Zetsche B. et al., Cell, 163(3):759-71, (2015)), and Target-AID (K. Nishida et al., Targeted nucleotide editing using hybrid prokaryotic and Examples include methods that use guide RNA and protein complexes, such as those described in "Vertebrate Adaptive Immune Systems, Science, DOI: 10.1126 / science.aaf8729, (2016)," or protein complexes.

[0035] The "Cas enzyme" is not particularly limited and can be selected appropriately depending on the purpose, and examples thereof include type I CRISPR enzymes, type II CRISPR enzymes, and type III CRISPR enzymes, with the type II CRISPR enzyme Cas9 being preferred. The "Cas9" is not particularly limited and can be selected appropriately depending on the purpose, and examples thereof include Streptococcus pneumoniae Cas9, Streptococcus pyogenes Cas9, Streptococcus thermophilus Cas9, and Staphylococcus aureus Cas9, with Streptococcus pyogenes Cas9 (SpCas9) being preferred. Alternatively, it may be a mutant of Cas9 derived from these organisms, or the D10A mutant of Cas9, which is known to function as a nickase (a DNA cleaving enzyme that nicks only one DNA strand), or 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 with a chemical mutagen (see, for example, Zwar and Chandler, Planta, 1995, vol. 197, pp. 39-48). Chemical mutagens are not particularly limited, but 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, or the like 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. Such known methods include, for example, DNA sequencing (next-generation sequencing, etc.), PCR, microarray analysis, and Southern blotting. Using these 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 portion thereof can be amplified by PCR, and then individuals having a mutation in the amplified product can be selected by the aforementioned TILLING 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 with 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 mutated GABA-T gene regulatory sequence are selected from the F1 plants. In this case, "a homozygote plant having the mutated GABA-T gene regulatory sequence" includes not only plants having two identical mutated GABA-T gene regulatory sequences (alleles), but also plants having a first GABA-T gene regulatory sequence having a first mutation and a second GABA-T gene regulatory sequence 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] The DNA or a vector into which the DNA has been inserted can be introduced into plant cells by various methods known to those skilled in the art, 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; for example, the above-mentioned site-specific nucleases, fusion proteins, and transposons can be introduced as proteins, and the above-mentioned guide RNA can be introduced as RNA.

[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, the 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, which contains 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, the vector into which the DNA has been inserted, the protein, and the RNA.

[0048] Such a pharmaceutical preparation may be one in which the two active ingredients are contained in a single composition, or may be one 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 methods described above. 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 with improved GABA concentrations can be obtained from the plant obtained in this manner. Therefore, the present invention also provides a method for producing edible parts with improved GABA concentrations, which includes the steps of producing a plant with improved GABA concentrations 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 dwarfism, 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, "dwarfism" means that the plant matures while remaining small compared to the plant before the introduction of the mutation in the present invention, and "no dwarfism" may also include a state in which the degree of dwarfism 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, callus, protoplasts, etc.) can be obtained from the plant or 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, callus, and protoplasts.

[0054] Furthermore, in the sexual reproduction described above, crossing with other lines (other varieties, etc.) can further enhance the GABA concentration and also produce offspring endowed with other traits (e.g., disease resistance, herbicide resistance, pest resistance, improved taste, improved storability, improved yield). To further enhance the GABA concentration, for example, crossing with a plant in which the function of the C-terminal autoinhibitory domain in GAD has been lost can be used, as shown in the Examples below. By crossing, a mutation can be artificially introduced into the regulatory sequence located upstream of the GABA-T gene, suppressing the expression of the gene and causing the function of the C-terminal autoinhibitory domain in GAD, thereby producing a plant with an improved GABA concentration.

[0055] In the present invention, "glutamate decarboxylase (GAD)" refers to an enzyme (registered under EC number 4.1.1.15) that catalyzes the conversion of glutamate to GABA. Examples include proteins belonging to the "Glutamate decarboxylase" category in UniProt and proteins encoded by genes belonging to the "GAD" category. A specific example is the tomato protein identified by UniProt accession number B1Q3F2. Table 2 lists the GAD genes from various plants, along with their respective registration IDs (gene IDs) in reference databases. The "C-terminal autoinhibitory domain" of GAD, which is subject to loss of function, is a calmodulin-binding domain (CaM BD) consisting of approximately 30 amino acids (e.g., 22-25 amino acids) located at the C-terminus, and functions as an autoinhibitory (autoinhibitory) domain that inactivates the enzymatic activity of GAD. Examples of such loss of function include 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 (e.g., Non-Patent Document 3).

[0056] [Table 2]

Example

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

[0058]

Table 3

[0059] <Genome editing targeting the upstream sequence of the GABA metabolism-related gene> As the test plant, Micro-Tom, a dwarf model variety of tomato (Solanum lycopersicum L.), was used. Using Sol Genomics Network (https: / / solgenomics.net), the genomic sequence of 2500 bases upstream from the start codon of the 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). For transformation, the Agrobacterium method optimized for Micro-Tom (Sun et al., Plant cell pysiology, 2006; 47(3): 426-431) was used, and Agrobacterium introduced with the above vector was infected into the cotyledon explants 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 selection media containing kanamycin were primarily selected as candidates for transformants.

[0061] Subsequently, to confirm that it is a transformant, genomic DNA was extracted from tomato young leaves, and primers specific to the CRISPR / Cas9 vector (SEQ ID NO: 6 and 7) were used to confirm whether the CRISPR / Cas9 vector was introduced into the tomato nuclear genome.

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

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

[0064] <Analysis of GABA metabolism-related gene genome editors> Next, to increase the variation of mutant alleles, one line of T0 individuals carrying the CRISPR / Cas9 vector was crossed with wild-type Micro-Tom or Micro-Tom SlGAD3 mutants in which the GABA accumulation in fruits was improved by genome editing of SlGAD3. The F1 lines obtained by crossing were further self-propagated, and null segregants that had mutations homozygously and lacked the CRISPR / Cas9 vector were obtained in their progeny (Figure 2).

[0065] In the SlGAD3 mutant, the C-terminal autoinhibitory domain of GAD3 was 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 set forth in SEQ ID NO: 14.

[0066] Next, 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. GABA content was measured using a GABA measurement kit (Enzyme Sensor Co., Ltd.). The method followed the instructions in the kit manual. Furthermore, the expression of the SlGABA-T1 gene was analyzed using qRT-PCR, with SlGABA-T1-specific primers (SEQ ID NOs: 12 and 13).

[0067] We then analyzed a line (Figure 3, hereafter referred to as "SlGAD3ΔC+SlGABA-T1Δpro") lacking 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 nucleotide sequence of SEQ ID NO: 1): 367 bases) in the SlGAD3ΔC background. SlGABA-T1 gene expression was suppressed in fruit (Figure 4, 79% reduction at the immature stage, 66% reduction at the chromatin break, and 33% reduction at the red ripening stage compared to the wild type). Furthermore, GABA accumulation in fruit was increased (Figure 5, 13-fold increase over the wild type and 2-fold increase compared to SlGAD3ΔC at the red ripening stage). However, no dwarfism or sterility was observed in the SlGAD3ΔC+SlGABA-T1Δpro line (Figure 6). [Industrial Applicability]

[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 having an improved concentration of gamma-aminobutyric acid (GABA), (1) 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; (2) A production method comprising a step of regenerating a plant from the plant cells in which expression of the gene has been suppressed in the 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 production method according to claim 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.

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

5. 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 claims 1 to 4, and obtaining an edible part from the plant body.

6. 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.

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 the plant according to any one of claims 6 to 9.

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