Genome-edited plant body

Abstract

The present invention provides a plant body having improved growth ability.　Provided is a genome-edited plant body that includes a base insertion and / or base deletion mutation in a promoter of the AHA2 gene or an ortholog gene thereof, wherein the level of the AHA2 protein or the ortholog protein thereof in the leaves is improved by the mutation.

Description

Genome-edited plants

[0001] The present invention relates to genome-edited plants and the like.

[0002] Plants must contend with a variety of environmental changes, including fluctuations in light intensity, water availability, temperature, and nutrient levels. One of the key components that controls both plant growth and productivity is the plasma membrane (PM) H. + PM H is an ATPase that plays a key role in many physiological processes, including cell elongation, nutrient uptake in roots, and light-induced stomatal opening. + The β-ATPase activity is regulated at multiple levels, including transcription, translation, and post-translational mechanisms. Phosphorylation of the penultimate amino acid residue, Thr, creates a binding site for 14-3-3 proteins, thereby activating PM H. + It is well known that PM H enhances the activity of ATPase. + The H-ATPase is important for light-induced stomatal opening. In the guard cells of Arabidopsis and poplar, plasma membrane H-ATPase was expressed using a strong promoter in guard cells by conventional transgenic methods. + Overexpression of the α-ATPase, AHA2, significantly enhances photosynthetic activity and plant growth (Non-Patent Document 1). However, gaining social consensus on genetically modified organisms (GMOs) produced by conventional genetic engineering approaches remains challenging. Therefore, it is essential to develop new strategies to address these issues.

[0003] The emergence of CRISPR / Cas9-mediated genome editing technology has greatly enhanced the potential for accelerating the breeding of non-genetically modified crops. Plant growth and productivity can be significantly improved by modulating cis-regulatory elements (CREs) in the promoters of genes with important functions. However, controlling gene expression levels is challenging due to influencing factors such as chromosomal interactions, epistasis, and inter-module compensation, and the inherent modularity and redundancy of CREs make it difficult to identify efficient targets.

[0004] Wang Y, Noguchi K, Ono N, Inoue SI, Terashima I, Kinoshita T. (2014) Overexpression of membrane plasma H+-ATPase in guard cells promotes light-induced stomatal opening and enhances plant growth. Proc Natl Acad Sci 111: 533-538.

[0005] An object of the present invention is to provide a plant with improved growth potential.

[0006] In view of the above-mentioned problems, the present inventors have conducted extensive research and found that the above-mentioned problems can be solved by a genome-edited plant that contains a base insertion and / or base deletion mutation in the promoter of the AHA2 gene or its orthologous gene, and that the level of the AHA2 protein or its orthologous protein in the leaves is improved by said mutation. Based on this finding, the present inventors have conducted further research and completed the present invention. That is, the present invention encompasses the following aspects.

[0007] Item 1. A genome-edited plant comprising a base insertion and / or base deletion mutation in the promoter of an AHA2 gene or an orthologous gene thereof, and wherein the level of an AHA2 protein or an orthologous protein thereof in leaves is improved by the mutation.

[0008] Item 2. The genome-edited plant according to Item 1, wherein the mutation is present within 4000 b upstream of the start codon of the AHA2 gene or an orthologous gene thereof.

[0009] Item 3. The genome-edited plant according to Item 1 or 2, wherein at least one of the mutations is present within 500 b upstream of the start codon of the AHA2 gene or an orthologous gene thereof.

[0010] Item 4. The genome-edited plant according to any one of Items 1 to 3, wherein the number of mutation sites is three or more.

[0011] Item 5. The genome-edited plant according to any one of Items 1 to 4, wherein the mutation occurs at positions 3 to 5.

[0012] Item 6. The genome-edited plant according to any one of Items 1 to 5, wherein the mutation is present within or near a conserved region of the promoter of the AHA2 gene or an orthologous gene thereof.

[0013] Item 7. The genome-edited plant according to Item 6, wherein the vicinity of the conserved region is within 200 b from the end of the conserved region.

[0014] Item 8. The genome-edited plant according to Item 6 or 7, wherein the conserved region is at least one region selected from the group consisting of within 300 b upstream of the start codon of the AHA2 gene or its orthologous gene, 1000 to 1500 b upstream of the start codon of the AHA2 gene or its orthologous gene, 1800 to 2300 b upstream of the start codon of the AHA2 gene or its orthologous gene, and 3000 to 3500 b upstream of the start codon of the AHA2 gene or its orthologous gene.

[0015] Item 9. The genome-edited plant according to any one of Items 1 to 8, wherein the level of the AHA2 protein or its orthologous protein is increased by 1.1 times or more.

[0016] Item 10. The genome-edited plant according to any one of Items 1 to 9, which is a Brassicaceae plant.

[0017] Item 11. A seed produced from the genome-edited plant according to any one of Items 1 to 10.

[0018] Item 12. A method for producing a genome-edited plant according to any one of Items 1 to 10, comprising the steps of: performing genome editing to introduce a base insertion and / or base deletion mutation into the promoter of the plant AHA2 gene or its orthologous gene; and selecting plants using the level of AHA2 protein or its orthologous protein in leaves as an indicator.

[0019] Item 13. A method for screening for plants with improved growth potential due to improved rhizosphere acidification ability and / or nitrogen absorption ability by roots, comprising the steps of performing genome editing to introduce a base insertion and / or base deletion mutation into the promoter of the plant AHA2 gene or its orthologous gene, and selecting plants using the level of AHA2 protein or its orthologous protein in leaves as an indicator.

[0020] According to the present invention, a plant with improved growth potential can be provided. Since the plant of the present invention is obtained by genome editing, it can be widely used without the limitations of genetically modified plants. Furthermore, the plant of the present invention not only has an improved leaf stomatal aperture rate, but also has improved rhizosphere acidification ability and / or nitrogen absorption ability by roots.

[0021] AHA2 transcription and translation levels in nine Cas9-free homozygous mutants. (A) Control (background plant) and AHA2 CR-pro Schematic diagram of the promoter region in the mutant. Deletions (-) and insertions (+) are indicated by numbers and letters. Blue arrows indicate sgRNAs, open regions indicate deletions, and open regions indicate insertions. (B) Quantitative real-time PCR analysis of AHA2 expression in roots and leaves of control and homozygous mutant plants. ACT2 and TUB2 were used as endogenous control genes. Error bars represent significant differences (n=3). (C) mVISTA plot of the AHA2 promoter and promoters of AHA2 orthologs from three Brassicaceae species. Gray shading indicates conserved regions (CR). Red regions indicate high sequence similarity (>70%) between A. thaliana and A. Lin; Alyssum linifolium; C. Amp; Caulanthus amplexicaulis; and C. gra; Capsella grandiflora. CR-pro Relative AHA2 protein levels in roots (D) and leaves (E) of homozygous mutants. Values ​​are the mean ± SE of three biological replicates. (F) PM H in control and homozygous mutant leaves. +-ATPase activity. Error bars represent SE (n=3). Two-tailed Student's t-test was used to detect significant differences (* P<0.05; ** P<0.01; *** P<0.001; ns, not significant). Control and AHA2 CR-pro Rhizosphere acidification and nitrate uptake from roots of homozygous mutants. (A) Control and AHA2 CR-pro Monitoring of rhizosphere acidification around the roots of the mutants. Analysis was performed using solid medium containing 0.02% bromocresol purple (pH indicator) and 0.8% agar at pH 6.5. 10-day-old control and AHA2 mutants were washed with deionized water. CR-pro (B) Mutant roots were carefully placed in the medium, incubated for 6 hours, and then photographed. The yellow indicates the acidified area. CR-pro Relative acidification area in the rhizosphere of the mutants. Values ​​are mean ± SE (n = 4–5). (c) 4-week-old control and AHA2 CR-pro The roots of the mutant were treated with vanadate (PM H + Nitrogen absorption rate (with or without ATPase inhibitor) 15 N). Small circles indicate the results of three independent experiments. Values ​​are means ± SE (n = 3). Differences were calculated using a two-tailed Student's t-test (*P < 0.05; **P < 0.01, ns indicates no significant difference). Control and AHA2 CR-pro Stomatal and photosynthetic characteristics of homozygous mutants. (A) qRT-PCR assay of AHA2 in guard cell-enriched epidermal fragments from 4-week-old plants. Data are means ± SE (n = 6). Small circles represent individual data points. Differences were assessed using Student's t test (***P < 0.001). (B) Stomatal and photosynthetic characteristics of homozygous mutants in the dark or light (50 μmol m -2 s -1 red light and 10 μmol m -2 s -1 Epidermal guard cells under blue light + (C) Representative image of an inflorescence showing immunohistochemical staining of PM H-ATPase. The white arrow indicates the location of stomata in the epidermal guard cells. +(D) Signal intensity of AHA-ATPase. Mean ± SE (n = 47–111). Small circles indicate individual data points. Differences were evaluated by Student's t-test (***P<0.01; ***P<0.001). (E) Control and AHA2 cells exposed to white light for 30 minutes. CR-pro-1 (E) Stomatal opening phenotypes in response to light or light and ABA treatment. Epidermal tissue from dark-adapted plants in stomatal opening buffer was either kept in the dark for 2.5 h or exposed to light (10 μmol m -2 s -1 50 μmol m in blue light -2 s -1 The cells were exposed to light (with red light superimposed) for 2.5 hours, or in the presence of 20 μM ABA. Data are means ± SE (n = 60). Differences were evaluated by Student's t-test (***P < 0.001). CR-pro Stomatal conductance (F) and CO2 assimilation rate (G) of the mutants. Measurements were taken at 750 μmol m -2 s -1 The experiments were performed under white light. Each independent experiment is represented by a small circle. Data are shown as mean ± SE (n = 3). Blue arrows indicate the lighting time. CR-pro Plant growth and flowering time of homozygous mutants. (A) 25-day-old control and AHA2 CR-pro Representative images of mutant phenotypes. Scale bar = 1 cm. (B) 25-day-old control and AHA2 CR-pro Aboveground shoot biomass of mutants. Data are means ± SE (n = 6). The significance of differences was determined by Student's t-test (***P < 0.01; ***P < 0.001; ns = not significant). (C) Cotyledon diameter of 7-day-old plants grown on solid medium containing 0.5% (w / v) sucrose. Data are means ± SE (n = 12–22). The significance of differences was determined by Student's t-test (*P < 0.05; **P < 0.01; ns = not significant). (D) Nitrogen (NO3 - ) or phosphorus (PO4 3-The primary root length of 7-day-old plants grown on solid medium containing 1 / 2 Hoagland nutrient solution with or without AHA2. Data are shown as mean ± SE (n = 11-29). The significance of differences was determined by Student's t-test (*P < 0.05; ns = not significant). (EF) Control and AHA2 CR-pro Number of days to flowering and number of rosette leaves at flowering in the mutants. Values ​​are means ± SE (n = 8). Small circles indicate individual data points. The significance of differences was determined by Student's t-test (*P < 0.05; **P < 0.01; ***P < 0.001; ns = not significant). Control and AHA2 CR-pro Agronomic traits and seed production of homozygous mutants. (A) 46-day-old control and AHA2 CR-pro Representative images of mutant phenotypes. Scale bar = 5 cm. (B) 78-day-old control and AHA2 CR-pro Representative images of seed phenotypes from three mutant individuals. (C) 46-day-old control and AHA2 mutants grown in summer and autumn. CR-pro Caryopsis number of mutants. Values ​​are mean ± SE (n = 6). Small circles indicate data points for each experiment. Significant differences were analyzed using Student's t-test (*P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant). (D) 46-day-old control and AHA2 mutants. CR-pro (E) Fresh weight per spore of mutants. Values ​​are means ± SE (n = 6). Small circles represent individual data points. Student's t-test was used to analyze differences (ns, not significant). (F) Number of branches per plant. Values ​​are means ± SE (n = 6). Small circles represent individual data points. CR-pro Seed weight of mutants. Values ​​are means ± SE (n = 6). Small circles represent data points from individual experiments. The significance of differences was determined by Student's t-test (*P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant). The Arabidopsis AHA2 promoter sequence (SEQ ID NO: 1) is shown. The cis-regulatory elements and gRNA target sites within the sequence are indicated.

[0022] In this specification, the expressions "contain" and "comprise" include the concepts of "contain," "comprise," "consist essentially of," and "consist only of."

[0023] "Identity" of amino acid sequences refers to the degree of correspondence between the amino acid sequences of two or more comparable amino acid sequences. Therefore, the greater the correspondence between two amino acid sequences, the greater the identity or similarity between those sequences. The level of identity of amino acid sequences can be determined, for example, using the sequence analysis tool FASTA with default parameters. Alternatively, it can be determined using the BLAST algorithm by Karlin and Altschul (Karlin S, Altschul SF. "Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes," Proc Natl Acad Sci USA. 87:2264-2268 (1990); Karlin S, Altschul SF. "Applications and statistics for multiple high-scoring segments in molecular sequences," Proc Natl Acad Sci USA. 90:5873-7 (1993)). A program called BLASTP, based on the BLAST algorithm, has been developed. Specific techniques for these analysis methods are known and can be found on the National Center of Biotechnology Information (NCBI) website (http: / / www.ncbi.nlm.nih.gov / ). The "identity" of nucleotide sequences is also defined in the same manner as above.

[0024] As used herein, nucleotide / amino acid mutations include nucleotide / amino acid substitutions, deletions, additions, insertions, etc. Amino acid mutations are preferably amino acid substitutions, more preferably conservative substitutions.

[0025] As used herein, the term "conservative substitution" refers to the substitution of an amino acid residue with an amino acid residue having a similar side chain. For example, substitution between amino acid residues having basic side chains such as lysine, arginine, and histidine constitutes a conservative substitution. Other examples of conservative substitutions include substitution between amino acid residues having acidic side chains such as aspartic acid and glutamic acid; amino acid residues having uncharged polar side chains such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues having nonpolar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues having β-branched side chains such as threonine, valine, and isoleucine; and amino acid residues having aromatic side chains such as tyrosine, phenylalanine, tryptophan, and histidine.

[0026] As used herein, the terms "nucleic acid" and "polynucleotide" are not particularly limited and encompass both natural and artificial nucleic acids. Specifically, in addition to DNA, RNA, and the like, known chemical modifications may be used, as exemplified below. To prevent degradation by hydrolases such as nucleases, the phosphate residue of each nucleotide may be substituted with a chemically modified phosphate residue, such as phosphorothioate (PS), methylphosphonate, or phosphorodithioate. Furthermore, the hydroxyl group at the 2-position of the sugar (ribose) of each ribonucleotide may be substituted with -OR (where R represents, for example, CH3(2'-O-Me), CH2CHOCH3(2'-O-MOE), CH2CH2NHC(NH)NH2, CH2CONHCH3, or CH2CH2CN). Furthermore, the base moiety (pyrimidine or purine) may be chemically modified, for example, by introducing a methyl group or a cationic functional group into the 5-position of the pyrimidine base, or by substituting a thiocarbonyl group for the carbonyl group at the 2-position. Further examples include, but are not limited to, those in which the phosphate moiety or hydroxyl moiety is modified with, for example, biotin, an amino group, a lower alkylamine group, an acetyl group, etc. Also usable are BNA (LNA), in which the conformation of the sugar moiety of the nucleotide is fixed to N-type by bridging the 2' oxygen and 4' carbon of the sugar moiety.

[0027] As used herein, the term "gene" includes not only the regions (exons, introns) on genomic DNA that code for a protein, but also the regions that control the expression of the protein (expression control regions).

[0028] In one aspect, the present invention relates to a genome-edited plant (sometimes referred to herein as the "genome-edited plant of the present invention") that contains a base insertion and / or base deletion mutation in the AHA2 gene promoter, and that has an improved AHA2 protein level in its leaves due to the mutation. This is described below.

[0029] The plant from which the genome-edited plant of the present invention is derived (non-genome-edited plant) is not particularly limited. Examples of plants include a wide range of plants, including angiosperms such as Magnolias, monocotyledons, and eudicotyledons (Rosaceae I, Rosaceae II, Chrysanthemums I, Chrysanthemums II, and their outgroups). Cultivars are particularly preferred. More specific examples of cultivated varieties include eggplants such as tomatoes, bell peppers, chili peppers, and eggplants; melons such as cucumbers, pumpkins, melons, and watermelons; vegetables such as cabbage, broccoli, and Chinese cabbage; fresh or spicy vegetables such as celery, parsley, and lettuce; onions such as leeks, onions, and garlic; beans such as soybeans, peanuts, green beans, peas, and adzuki beans; other fruit vegetables such as strawberries; taproots such as radishes, turnips, carrots, and burdock; potatoes such as taro, cassava, potato, sweet potato, and Chinese yam; soft vegetables such as asparagus, spinach, and mitsuba; flowers such as lisianthus, stock, carnations, and chrysanthemums; rice, wheat, barley, oats, and corn. Examples of suitable plants include cereals, grasses such as bentgrass and Zoysiagrass, oil crops such as rapeseed and peanut, sugar crops such as sugarcane and sugar beet, fiber crops such as cotton and rush, forage crops such as clover, sorghum and dent corn, deciduous fruit trees such as apples, pears, grapes and peaches, citrus fruits such as satsuma mandarins, lemons and grapefruits, woody plants such as azalea, azalea and cedar, and nuts and seeds such as almonds, hemp, flax, perilla, cashew, pumpkin, Japanese kaya, ginkgo, chestnut, walnut, poppy, coconut, sesame, Japanese castanea, watermelon, chia, horse chestnut, lotus, water chestnut, pistachio, sunflower, Brazil nut, hazel, pecan, macadamia, pine and peanut. In one embodiment of the present invention, Brassicaceae plants are preferred among these.

[0030] The plant body means the entire plant including all of the plant tissues (roots, stems, leaves, etc.).

[0031] The AHA2 gene, which is the target of modification, is located in the plasma membrane (PM) H of Arabidopsis thaliana. +The AHA2 gene encodes the AHA2-ATPase (NCBI Gene ID: 829142). The coding sequence of the AHA2 gene is the nucleotide sequence shown in SEQ ID NO: 2, and the amino acid sequence of the AHA2 protein is the amino acid sequence shown in SEQ ID NO: 3.

[0032] The orthologous gene of the AHA2 gene to be modified can be easily identified based on a homology search using the sequence information of the AHA2 gene and also based on publicly known findings. In one embodiment, the protein encoded by the orthologous gene has an amino acid sequence that is 80% or more identical (preferably 85% or more, more preferably 85% or more, even more preferably 90% or more, even more preferably 95% or more, particularly preferably 98% or more, and particularly preferably 99% or more) to the amino acid sequence shown in SEQ ID NO: 3, and has a PM H + AHA2 is a protein with α-ATPase activity. Examples of orthologous genes of the AHA2 gene include rice (Oryza sativa) OSA1 (NCBI Gene ID: Q43001).

[0033] PM H + The -ATPase activity was measured using the method described below in the "Methods and Materials" section of the Examples. + -Measurement of ATP hydrolysis activity of ATPase>.

[0034] The AHA2 gene or its orthologous gene to be modified is a gene inherent to the target plant of genome editing. When the target plant is Arabidopsis thaliana, the modification target is the AHA2 gene, and when the target plant is another plant, the modification target is the AHA2 gene ortholog in that plant.

[0035] The AHA2 gene or its orthologous gene to be modified also includes functionally normal mutants that may occur in nature.

[0036] The promoter of the AHA2 gene or its orthologous gene is a transcriptional regulatory region upstream of the start codon of the AHA2 gene or its orthologous gene, and is not particularly limited as long as it is such a region. The promoter is, for example, 10,000 bases, preferably 8,000 bases, more preferably 6,000 bases, and even more preferably 4,000 bases upstream of the start codon of the AHA2 gene or its orthologous gene.

[0037] The genome-edited plant of the present invention contains a mutation in the promoter, such as a base insertion and / or base deletion, which mutation is preferably located within 4000 b upstream of the start codon of the AHA2 gene or its orthologous gene.

[0038] The number of mutation sites in the promoter is, for example, 2 or more, preferably 3 or more, more preferably 3 to 6, even more preferably 3 to 5, and even more preferably 3 to 4. The more such sites there are, the more effectively the expression level of the AHA2 gene or its orthologous gene can be regulated, and the risk of off-target effects can be further reduced by keeping the number of such sites below a certain level.

[0039] At least one of the mutations is preferably located near the start codon where a relatively large number of transcriptional regulatory regions are present, particularly within 500 bp (more preferably within 400 bp, and even more preferably within 300 bp) upstream of the start codon of the AHA2 gene or its orthologous gene, which allows for more effective regulation of the expression level of the AHA2 gene or its orthologous gene.

[0040] It is desirable that the number of inserted or deleted bases in the mutation be small. This number is preferably 50 or less, more preferably 30 or less, and even more preferably 20 or less. The mutation (especially a mutation near the start codon where a relatively large number of transcriptional regulatory regions are present) is preferably a single-base insertion and / or a single-base deletion. Mutations other than single-base insertion and single-base deletion are preferably at two or less locations, particularly preferably at one or less location. This allows for more effective regulation of the expression level of the AHA2 gene or its orthologous gene.

[0041] The mutation is preferably located within or near a conserved region of the promoter of the AHA2 gene or its orthologous gene.

[0042] A conserved region is a region that is highly conserved within plants of the same family. Specifically, the promoter sequence is compared with that of three different plant species of the same family, analyzed according to the method described in "Analysis of AHA2 promoter sequences in the Brassicaceae family" in the "Methods and Materials" section of the Examples below, and determined to be a conserved region when the similarity threshold is set at 70%.

[0043] The vicinity of the conserved region means, for example, within 200 b, preferably within 150 b, from the end of the conserved region.

[0044] The conserved region is, for example, at least one region selected from the group consisting of within 300 b upstream of the start codon of the AHA2 gene or its orthologous gene, 1000 to 1500 b upstream of the start codon of the AHA2 gene or its orthologous gene, 1800 to 2300 b upstream of the start codon of the AHA2 gene or its orthologous gene, and 3000 to 3500 b upstream of the start codon of the AHA2 gene or its orthologous gene.

[0045] It is preferable that at least one of the mutations is located within or near 300 b upstream of the start codon of the AHA2 gene or its orthologous gene, and it is particularly preferable that the mutations are located in at least two regions selected from the group consisting of 1000 to 1500 b upstream of or near the start codon of the AHA2 gene or its orthologous gene, 1800 to 2300 b upstream of or near the start codon of the AHA2 gene or its orthologous gene, and 3000 to 3500 b upstream of or near the start codon of the AHA2 gene or its orthologous gene.

[0046] The above mutations are designed to avoid disruption of the core CRE, including the TATA-box and CAAT-box.

[0047] The above mutations are introduced by genome editing.

[0048] The genome-edited plant of the present invention preferably has the above mutation in both of the paired chromosomes.

[0049] In the genome-edited plant of the present invention, the above mutation is preferably present in all cells of the plant.

[0050] The genome-edited plant of the present invention has an improved level of AHA2 protein or its orthologous protein in its leaves due to the above mutation. The present inventors have discovered that in genome-edited plants obtained by genome-editing the promoter of the AHA2 gene or its orthologous gene, using the level of AHA2 protein or its orthologous protein in the leaves as an indicator not only improves the leaf stomatal aperture rate but also, unexpectedly, improves the rhizosphere acidification ability and / or nitrogen absorption ability of the roots, thereby completing the present invention.

[0051] The "level" refers to the amount of expression. The level of the AHA2 protein or its orthologous protein in leaves can be measured according to the description regarding Western blot in the "Immunoblot Assay" section of the "Methods and Materials" section of the Examples below.

[0052] In the genome-edited plant of the present invention, the level of AHA2 protein or its orthologous protein is preferably increased by 1.1 times or more due to the above mutation.

[0053] In one aspect, the present invention relates to a seed generated in the genome-edited plant of the present invention. The seed can be obtained, for example, from the genome-edited plant of the present invention having the above-mentioned mutation in a germ cell.

[0054] The genome-edited plant of the present invention can be obtained by a method comprising the steps of performing genome editing to introduce a base insertion and / or base deletion mutation into the promoter of the plant AHA2 gene or its orthologous gene, and selecting plants using the level of AHA2 protein or its orthologous protein in leaves as an indicator. From this perspective, in one aspect, the present invention relates to a method for producing the genome-edited plant of the present invention, and a method for screening plants having improved rhizosphere acidification ability and / or nitrogen absorption ability by roots and therefore improved growth potential, which comprise these steps, as will be described below.

[0055] Specific examples of methods for introducing mutations include introducing into plant cells an insert containing at least one selected from the group consisting of a target-specific nuclease, an expression cassette for the nuclease, and mRNA for the nuclease. Cleavage of the target site by the target-specific nuclease and subsequent repair results in the introduction of base insertion and / or base deletion mutations at the target site.

[0056] The target-specific nuclease is not particularly limited as long as it is a nuclease that can specifically cleave a specific site on genomic DNA to induce a mutation. Examples of target-specific nucleases include Cas proteins, TALEN proteins, and ZFN proteins.

[0057] The CRISPR / Cas system uses a Cas protein, a nuclease (RGN; RNA-guided nuclease), and a guide RNA. By introducing this system into cells, the guide RNA binds to the target site, and the Cas protein is recruited to the binding site and can cleave the DNA.

[0058] The TALEN system using TALEN protein uses artificial nuclease (TALEN) that comprises DNA cleavage domain (for example, FokI domain) and the DNA binding domain of transcription activator-like (TAL) effector.By introducing this system into cells, TALEN binds to target site through DNA binding domain and cuts DNA there.The DNA binding domain that binds to target site can be designed according to known schemes (for example, Zhang F et al. (2011) Nature Biotechnology 29 (2); this paper is incorporated herein by reference).

[0059] The ZFN system using ZFN protein uses artificial nuclease (ZFN) that comprises nucleic acid cutting domain conjugated with DNA binding domain that comprises zinc finger array.By introducing this system into cells, ZFN binds to target site through DNA binding domain and cuts DNA there.The DNA binding domain that binds to target site can be designed according to known scheme.

[0060] Among target-specific nucleases, Cas proteins are preferred from the viewpoint of being able to more freely determine the cleavage site, and a preferred example of the Cas protein is the Cas9 protein.

[0061] The target-specific nuclease expression cassette is not particularly limited as long as it is DNA capable of expressing a target-specific nuclease in the cells of the subject of the production method of the present invention. A typical example of a target-specific nuclease expression cassette is DNA comprising a promoter and a target-specific nuclease coding sequence placed under the control of the promoter. The target-specific nuclease expression cassette may comprise a vector alone or together with other sequences (e.g., a drug resistance gene, an origin of replication, etc.). The type of vector is not particularly limited.

[0062] When the target-specific nuclease is a Cas protein, the introduced material in the plant production method of the present invention further includes at least one selected from the group consisting of a guide RNA expression cassette and a guide RNA.

[0063] The guide RNA is not particularly limited as long as it is one that can be used in the CRISPR / Cas system. For example, various types of guide RNAs can be used that can bind to a target site in genomic DNA and bind to a Cas protein, thereby guiding the Cas protein to the target site in genomic DNA.

[0064] It is said that the 12 bases on the 3' side of the crRNA sequence that binds to the target sequence are important for the binding of the guide RNA to the target site. Therefore, if the crRNA sequence that binds to the target sequence is not completely identical to the target strand, it is preferable that the bases that differ from the target strand exist in the crRNA sequence other than the 12 bases on the 3' side of the sequence that binds to the target sequence.

[0065] The target of introduction is not particularly limited, and may be undifferentiated plant tissue (e.g., callus), a part of a seed (e.g., hypocotyl, shoot apex, etc.), or a part of an adult plant (e.g., shoot apex, etc.).

[0066] The introduction method is not particularly limited as long as it allows the introduced substance to reach the plant cells, and can be appropriately selected depending on the type of substance to be introduced and the target of introduction. Examples of introduction methods include the floral dip method, floral spray method, Agrobacterium method, particle gun method, infiltration method, toothpick inoculation method, suction injection method, leaf disc method, inflorescence infiltration method, vacuum filtration method, virus-mediated nucleic acid delivery, etc. Among these, the Agrobacterium method is preferred from the viewpoints of simplicity, safety, etc.

[0067] A more specific example of how to implement it is shown below.

[0068] A first specific example of the introduction method (Introduction Example 1) includes the steps of: preparing a plasmid containing a promoter (e.g., T7 promoter, T3 promoter, 35S promoter, etc.) and a sequence containing an expression cassette downstream of the promoter (step a1); obtaining plant virus genomic RNA from the plasmid obtained in step a1 by in vitro transcription (step b1); and inoculating a plant with the genomic RNA (active ingredient) obtained in step b1 (e.g., friction inoculation, particle gun inoculation, etc.) (step c1). Alternatively, if the plasmid obtained in step a1 is a Ti plasmid containing a promoter capable of activating transcription in plant cells, such as the 35S promoter, instead of steps b1 and c1, the introduction can be carried out by a method including, for example, introducing the plasmid obtained in step a1 into Agrobacterium and culturing it (step b2), and inoculating a plant with the culture solution obtained in step b2 (containing the active ingredient) (e.g., infiltration, toothpick inoculation, suction injection, etc.) (step c2). Alternatively, instead of steps b1 and c1 above, a method including a step (step c3) of inoculating a plant with the plasmid (active ingredient) obtained in step a1 (for example, grinding inoculation, particle gun inoculation, etc.) can be used. Alternatively, instead of step c2 above, a method including a step (step c4) of performing, for example, the leaf disc method, inflorescence infiltration method, vacuum filtration, etc. can be used. The desired protein or peptide is produced from the genomic RNA, plasmid, T-DNA, etc. introduced into the plant by these methods.

[0069] A second specific example of the introduction method (Introduction Example 2) includes a step (step d1) of collecting a plant virus from a plant containing the virus (e.g., obtained by the above-mentioned Introduction Example 1), and a step (step e1) of inoculating a plant with the virus (a virus containing an active ingredient) collected in step d1. The collection in step d1 can be carried out, for example, by grinding a part of the plant containing the plant virus (e.g., a leaf) and recovering the virus solution. The inoculation in step e1 can be carried out, for example, by using an abrasive such as silicon carbide to make a wound in the part of the plant to be inoculated (e.g., a leaf) and contacting the wound with the virus.

[0070] After the introduction, genome-edited plants can be obtained by growing the resulting plant or plant cells, or by growing the resulting plant through callus. Furthermore, after the introduction, the introduced cells, tissues, etc. can be selected with a drug, if necessary.

[0071] From the obtained genome-edited plants, genome-edited plants of the present invention are selected using the level of AHA2 protein or its orthologous protein in leaves as an indicator, which allows for efficient production of genome-edited plants that not only have improved leaf stomatal aperture but also, unexpectedly, improved rhizosphere acidification ability and / or nitrogen uptake ability by roots.

[0072] The above-mentioned introduced material can be used as an agent for producing a genome-edited plant of the present invention.

[0073] The manufacturing agent of the present invention may consist solely of the above-described introduced substance (essential component), but may also contain various other components in addition to the essential component, depending on the type of essential component contained, the dosage form (described below), and the mode of use. The content of the essential components (dry weight) in the manufacturing agent of the present invention can be determined appropriately depending on the dosage form (described below) and the mode of use, and can range, for example, from 0.0001 to 100% by mass. Examples of other components include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, humectants, colorants, fragrances, and chelating agents. The form of the manufacturing agent of the present invention is not particularly limited and can be, for example, a dry form, a solution form, or a kit form. The kit may also contain other materials, reagents, and tools necessary for plant production, such as nucleic acid transfer reagents and buffer solutions, as needed.

[0074] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0075] Methods and Materials The methods and materials used in the test examples described below are as follows.

[0076] <Plant cultivation> Arabidopsis thaliana (Columbia), which carries the gl1 (control) and homozygous recessive gl1 alleles, was used as the background ecotype for all mutants. For experiments involving stomatal aperture measurement, gas exchange, immunohistochemical detection, and biomass productivity, plants were grown in a growth chamber under 16 h of fluorescent light (50 μmol m ) at 24°C and 55–70% humidity (Pa / Pa). -2 s -1 Plants were grown in soil under a 10-hour light / 8-hour dark cycle. To measure cotyledon diameter and root length, plants were grown on solid medium containing 1 / 2 Hoagland's nutrient solution at pH 5.7, with or without nitrogen or phosphorus, under the same environmental conditions except for 100% humidity. Plants used for qRT-PCR assays and Western blots were grown hydroponically in 3-L containers containing 1 / 2 Hoagland's nutrient solution at pH 5.7, with the nutrient solution changed every 3 days.

[0077] <Design and Construction of sgRNA Vectors> Four sgRNAs were designed to recognize conserved or non-conserved regions in the promoter sequence of AHA2 from Arabidopsis thaliana (http: / / www.e-crisp.org). The vectors were constructed based on previous methods with minor modifications. The CRISPR / Cas9 expression vector was constructed using the pKRCAS9 vector, which contains attl1 / 2 sites driven by the highly constitutive RPS5A promoter. For the multi-gateway LR reaction, a binary vector containing various combinations of BbsI restriction enzyme sites and attl sites (attl1 / 5, attl5 / 4, attl4 / 3, attl3 / 2) driven by the Arabidopsis AtU6 promoter was used. The amplified 20-bp gRNAs were annealed and inserted into the BbsI site of the pEn-Chimera vector. Using multiple Gateway LR reactions, customized RNA chimeras (sgRNA expression cassettes) containing four specific target sites were inserted into pKRCAS9 in a single Gateway LR reaction to generate the pKRCAS9-pAHA2 plant expression vector.

[0078] Transformation, Genotyping, and Sequencing Analysis: pKRCAS9-pAHA2 was transformed into gl1 (control) by floral dipping using Agrobacterium tumefaciens GV3101. T0-positive transgenic seedlings were screened on antibiotic plates. T1 plants were identified by genotyping and Sanger sequencing. To select positive transfections in the T1 generation, PCR was performed using total DNA to amplify the T-DNA sequence (primers gRNA-F / PMR302-R and Kan-F / Kan-R). PCR products were electrophoresed on a 1% agarose gel. In the T2 and T3 generations, the transgene was identified using two pairs of Cas9 gene-specific primers, cas-167-F / cas-167-R and cas-471-F / cas-471-R, and the quality of the PCR product was confirmed using the control gene ARF6 (ARF6-F / ARF6-R). Mutations in the sgRNA target site of T3-positive transformants were confirmed by PCR using primers pAHA2-sq1-F / pAHA2-sq1-R, pAHA2-sq2-F / pAHA2-sq2-R, pAHA2-sq3-F / pAHA2-sq3-R, and pAHA2-sq4-F / pAHA2-sq4-R. The PCR products were then subjected to Sanger sequencing.

[0079] <Analysis of potential off-target sequences of sgRNAs> Potential off-target sites of gRNA1, gRNA2, gRNA3, and gRNA4 were analyzed using the online software benchling (https: / / benchling.com). To detect potential off-target sites in the T4 homozygous generation, PCR products encompassing potential off-target sites were subjected to Sanger sequencing using primers.

[0080] <Analysis of the AHA2 Promoter Sequence in the Brassicaceae Family> Conserved regions in the AHA2 promoter were identified. Three Brassicaceae species, Alyssum linifolium (Alyli.0150s0097), Caulanthus amplexicaulis (Caamp.1041s0429), and Capsella grandiflora (Cagra.2848s0018), were searched and phylogenetic footprinting was performed using the GoGE GEvo tool (Lyons E, and Freeling M. (2008), Plant J 53, 661-673.). Sequence homology with Arabidopsis AHA2 was scored using mVISTA with a 100-bp alignment window and a similarity threshold of 70% for the approximately 4,000 bp upstream of the AHA2 CDS (https: / / genome.lbl.gov / vista / index.shtml). Seven conserved regions were identified (Figure 1A).

[0081] Comparison of sgRNA-binding proteins between wild-type and novel alleles of the AHA2 promoter Using the Plant ChIP-seq Database (PCBase; http: / / pcbase.itps.ncku.edu.tw), we searched for transcription factors (TFs), histones, and other proteins that bind to the four gRNA target sites with similarity levels of approximately 83% to 100%. Visualization of this database using TBtools software (Chen et al., 2020) revealed numerous components within the four sgRNA target sites and the 4000-bp promoter region. Furthermore, all AHA2 CR-pro The novel alleles of the homozygous mutants created new TF binding sites for the relative components based on this database.

[0082] <qRT-PCR assay> gl1 and AHA2 were assayed using the RNeasy Plant Mini Kit (Qiagen). CR-proTotal RNA was extracted from guard cell-enriched epidermal sections, leaves, and roots of the mutant. 400 ng of RNA was reverse transcribed into cDNA using the PrimeScript II First Strand cDNA Synthesis Kit and oligo(dT) primers (Takara). Quantitative PCR was performed using the Power SYBR Green PCR Master Mix and the StepOne Real-Time PCR System (Applied Biosystems) as previously described. Three independent experiments were performed. Relative gene expression was determined by comparing cycle thresholds. ACT2 and TUB2 were used as internal controls to normalize the relative amounts of AHA2 PCR products.

[0083] <Immunoblot assay> Western blot was performed using a previously published method with some modifications. CR-pro 0.1-0.2 g of mutant leaves and roots were immediately homogenized in liquid N and suspended in 200 μl of ice-cold homogenization buffer (50 mM MOPS-KOH, pH 7.5, 5 mM EDTA, 100 mM NaCl, 0.5 mM PMSF, 10 μM leupeptin, 2 mM dithiothreitol [DTT], 10 mM NaF, and 0.5 mM ammonium molybdate). Samples were centrifuged at 10,000 x g for 1 minute at room temperature to remove cellular debris, and the protein content in the supernatant was measured using the Bio-Rad Protein Assay (BIO-RAD, USA). 20 μg of protein from leaves and 15 μg from roots were solubilized in sodium dodecyl sulfate (SDS)-polyacrylamide gel sample buffer. PM H + -ATPase protein is expressed by anti-H + The PM H-ATPase antibody was used to detect the enzyme. + Image J was used to analyze the -ATPase signal intensity.

[0084] Following the previous method, PM H in guard cells + Immunohistochemical detection of AHA-ATPase was performed in the AHA2 promoter-edited plants (AHA CR-pro) and dark-adapted epidermal tissue from mature rosette leaves of control plants was treated with 50 μmol m s -2 -1 of red light or 10 μmol m -2 s -1 The tissues were exposed to 10 ... A blocking solution containing 3% BSA (v / v) fraction V in PBS was applied to the samples for 1 h at room temperature. Primary antiserum (anti-PM H + The secondary antibody (Alexa Fluor 546-conjugated goat anti-rabbit IgG [Invitrogen; A11034]) was diluted 1:100 in blocking solution and incubated at 37°C overnight. The specimens were washed three times with PBS and covered with a coverslip soaked in 50% (v / v) glycerol. Fluorescence images were captured using a Hg arc lamp with a CCD camera system (DP72; Olympus) and processed with DP2-BSW software (Olympus). Fluorescence intensity was measured using ImageJ (NIH, Bethesda, MD, USA).

[0085] <Measurement of stomatal aperture> Stomatal characteristics were measured using a previous method (Wang et al.). Plants used for measuring stomatal aperture were cultured overnight in the dark. Four-week-old AHA2 CR-pro and control rosette leaves were exposed to white light (150 μmol m -2 s -1After 2.5 hours of illumination at approximately 9:00–10:00 a.m., stomatal apertures were measured using a microscope (BX43; Olympus). At least 60 stomatal apertures were counted for each observation.

[0086] <Gas exchange parameters> Stomatal conductance and CO2 assimilation rate were measured using the LI-6400 system (Li-Cor). The measurement conditions were leaf temperature, relative humidity, flow rate, and leaf size at 24°C, 40-50% (Pa / Pa), and 400 mol s -1 After 10 minutes of initial dark adaptation, the leaves were illuminated with 750 μmol m -2 s -1 Three biological replicates were performed for each treatment.

[0087] <Depending on the root 15 N absorption rate > Nitrogen isotopes were measured using a Flash Flash2000-DELTAplus Advantage ConFlo III System (Instrument; thermoFisher Scientific; Japan). 15 No. 3 - To measure the absorption rate, 4-week-old gl1 and AHA2 mice were CR-pro Plants were pre-incubated overnight in Milli-Q water. Seedlings were rinsed with 0.1 mM CaSO4 for 1 min and then added to 4 mM NaCl. 15 NO3 (atom%) 15 They were transferred to a modified 1 / 2 Hoagland nutrient solution containing N; 98%) and exposed to white light (∼150 μmol m -2 s -1 ) for 5 min and then rinsed again with 0.1 mM CaSO4 for 1 min. The roots were then separated and dried at 70°C for 48 h. Three biological replicates were performed for each experiment.

[0088] Detection of rhizosphere acidification: The rate of proton extrusion from Arabidopsis roots was detected using a pH indicator solution containing 0.02% (w / v) bromocresol violet (pH indicator) and 0.8% (w / v) agar adjusted to pH 6.5. Roots of 10-day-old plants washed with deionized water were carefully soaked with a pH indicator solution consisting of 1 / 2 Hoagland nutrient solution. The pH indicator solution was illuminated by white light (150 μmol m -2 s -1 ) for 3, 6, and 12 hours, and the relative H was visualized using ImageJ (NIH, Bethesda, MD, USA) with the acidification area normalized to a yellow background. + The extraction rate was calculated.

[0089] <PM H + Measurement of ATP hydrolysis activity of PM H-ATPase + The α-ATPase activity was measured as follows. CR-pro Mature rosette leaves from homozygous mutants were harvested at 9:30 AM, and microsomal membranes were isolated. 45 μl of microsomal protein (20 μg) was added to ATPase reaction buffer (6 mM MgSO, 60 mM Tris-MES [pH 6.5], 100 mM KCl, 200 mM KNO, 1 mM ammonium molybdate, 10 μg mL). -1 The mixture was mixed with 10 mM orthovanadate (0.1% [w / w] oligomycin, 0.1% [w / w] Triton X-100, 0.5 mM PMSF, and 10 μM leupeptin). To measure vanadate-sensitive ATP hydrolysis activity, 2 μL of 10 mM orthovanadate was added to the mixture or not. The reaction was initiated by adding 10 μL of 2 mM ATP and incubated at 30°C for 30 min. The reaction was then stopped by adding 1 mL of 1.3% (w / v) SDS, 0.25% (w / v) sodium molybdate, and 0.3 N H2SO4. Inorganic phosphate was measured at a wavelength of 750 nm (OD) using a spectrophotometer (UH5300; Hitachi, Ibaraki, Japan). 750 ). Experiments were repeated with 3 biological replicates.

[0090] Example 1: CRISPR / Cas9-mediated mutations in the AHA2 promoter generated novel cis-regulatory alleles. The AHA2 promoter was analyzed using the online tool Plant CARE (https: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ). The AHA2 promoter contains numerous conserved cis-regulatory elements (CREs) associated with gene expression and environmental responses, including the TATA-box (TATATA), CAAT-box (CCAAT), G-box (CACGTT), MYB motif, and MYC motif (Figure 6). The presence of numerous TATA-box sites (12 in total) in the AHA2 promoter suggests that the TATA-box may act as an activator of gene transcription and thus contribute to the high expression level of AHA2.

[0091] Next, we compared the AHA2 (AT4G30190) promoter with promoters of AHA2 orthologs from three Brassicaceae plants—Alyssum linifolium (Alyli.0150s0097), Caulanthus amplexicaulis (Caamp.1041s0429), and Capsella grandiflora (Cagra.2848s0018)—to identify conserved regions (Fig. 1C). Pairwise sequence alignment revealed the presence of seven conserved regions (CRs) shared by all orthologs, approximately 100 to 200 bp in size and with high sequence identity (>70%) (Fig. 1C). Consequently, we designed four single guide RNAs (sgRNAs) targeting both conserved and non-conserved regions of the AHA2 promoter for promoter editing, while avoiding disruption of the core CRE, including the TATA-box and CAAT-box (Fig. 1A, Fig. 6). The specificity of these sgRNAs for their target sequences was confirmed with a high score (https: / / www.benchling.com).These sgRNAs were then co-cloned into the pMR302 vector, a powerful tool for Arabidopsis genome engineering, and subjected to floral-dip transformation.

[0092] In the T1 generation, we identified 20 candidate plants from a total of 27 kanamycin-resistant plants that were confirmed to carry the desired transgene. Nucleotide sequences of the AHA2 promoter region of these 20 candidates were determined, and 14 plants showed mutations in the sgRNA target site. Notably, the editing efficiency in the T1 generation reached approximately 52%. To achieve Cas9-free homozygosity in the T2 generation, 6–9 plants from each T2 line were genotyped for the Cas9 transgene. Genomic PCR revealed that only approximately 3.2% of the T2 mutants were Cas9-free. Consequently, we further self-fertilized the T2 mutants. Ultimately, approximately 13 T3Cas9-free lines (T3-1, T3-3, T3-5, T3-9, T3-12, T3-13, T3-15, T3-16, T3-20, T3-21, T3-23, T3-25, and T3-26) were identified. Because most of the T1 mutants were heterozygous or biallelic, their descendants may have been re-edited with the Cas9 transgene. Therefore, it is necessary to identify homozygotes among the T3Cas9-free candidates.

[0093] Next, we performed genomic PCR to amplify the sgRNA target regions in T3 Cas9-free lines and analyzed their genotypes. We found that specific sgRNA target sites, such as sgRNA-S1, failed to amplify in T3-20 and T3-21 lines. This suggests the possibility of chromosomal structural changes, such as extensive DNA deletions, that prevent PCR amplification. Furthermore, a long insertion was observed in sgRNA-S2 in T3-20. Furthermore, 14 lines (T3-1b, T3-3a, T3-3b, T3-5a, T3-7b, T3-9a, T3-12b, T3-13c, T3-16b, T3-16c, T3-15b, T3-25c, T3-23b, and T3-26c) showed PCR product sizes comparable to those of the wild-type allele at four sgRNA target sites. (Note: 'b' and 'c' indicate individual plants for each line.) This suggests that these sites may be free of mutations or may contain small deletions or indels.

[0094] Sanger sequencing was performed to characterize the nucleotide sequences of the four gRNA target sites in the 14 aforementioned lines. T3-1b (AHA2 CR-pro-1 ), T3 -3a (AHA2 CR-pro-3 ), T3 -5a (AHA2 CR-pro-5 ), T3 -9a (AHA2 CR-pro-9 ), T -12b (AHA2 CR-pro-12 ), T -13c (AHA2 CR-pro-12 ), T3 -12b (AHA2 CR-pro-12 ), T3 -13c (AHA2 CR-pro-13 ), T3 -15b (AHA2 CR-pro-15 ), T3 -16b (AHA2 CR-pro-16 ), T3 -23b (AHA2 CR-pro-23 ), and T3 -25c (AHA2 CR-pro-25 ) (Figure 1). AHA2 CR-pro-25 Analysis of AHA2 was discontinued due to poor growth. CR-pro-7 , AHA2 CR-pro-20 , AHA2 CR-pro-21 , and AHA2 CR-pro-26 No homozygotes were obtained for the T3 AHA2 strain. CR-pro In homozygous mutants, AHA2 CR-pro-1 (gRNA-T2: 3 bp deletion and 19 bp insertion), AHA2 CR-pro-15 (gRNA-T3 with 18 bp deletion and 3 bp insertion), AHA2 CR-pro-16 (16 bp deletion in gRNA-T2), and AHA2 CR-pro-23 Most gRNA target sites showed deletions or insertions of 1-2 bp, except for gRNA-T1 (7 bp deletion and 6 bp insertion). CR-pro-3 and AHA2 CR-pro-23 was mutated at two sites, whereas the majority of T3 homozygous mutants had mutations at three or four target sites (Fig. 1).

[0095] Nine T3 homozygous mutants, namely AHA2 CR-pro-1 , AHA2 CR-pro-3 , AHA2 CR-pro-5 , A.H.A. 2CR-pro-9, AHA2 CR-pro-12 , AHA2 CR-pro-13 , AHA2 CR-pro-15 , AHA2 CR-pro-16 , and AHA2 CR-pro-23 To investigate the presence of CRISPR / Cas9 off-target mutations in Arabidopsis, we used the CRISPR-P online tool to predict putative off-targets for four sgRNAs on the Arabidopsis genome. A total of 13 off-target sites were predicted as potential off-targets (3 for sgRNA1, 2 for sgRNA2, 3 for sgRNA3, and 5 for sgRNA4). To evaluate the potential off-target effects of these sgRNAs, we used the AHA2 CR-pro We sequenced the putative off-target annotated genes with protospacer adjacent motifs (PAMs) in the homozygous plants. No mutations were found in any of these genes, indicating the high specificity of these sgRNAs designed to target the AHA2 promoter. Therefore, we decided to use these nine T3 homozygous mutants for further analysis.

[0096] Test example 2. AHA2 CR-pro The transcription and translation levels of AHA2 are altered in homozygous mutant strains. First, quantitative RT-PCR (qRT-PCR) analysis revealed that the Cas9-free AHA2 CR-pro The relative expression levels of AHA2 were examined in nine homozygous mutant lines. CR-pro-1 , AHA2 CR-pro-5 , AHA2 CR-pro-13 In the case of α-AHA2, the relative expression level of AHA2 was significantly increased by approximately 2-3 times in both leaves and roots. CR-pro-3 and AHA2 CR-pro-12 A significant decrease of approximately 40% to 52% was observed in the leaves and roots of AHA2 compared to the background plants (control) (Fig. 1B). CR-pro-15 In leaves, the relative expression level of AHA2 was approximately two-fold higher, whereas AHA2 CR-pro-9 and AHA2 CR-pro-23 In leaves, the levels were approximately 39% and 47% lower than in the control, but no significant difference was observed in roots (Fig. 1B).

[0097] AHA2 CR-pro The amount of PM H+-ATPase protein in the homozygous mutant strains was assessed by Western blot to evaluate the effect of the promoter mutation on the translation level of AHA2 (Fig. 1D). CR-pro-1 and AHA2 CR-pro-13 The H+-ATPase levels in roots of AHA2 were approximately 40% and 51% higher than those in the control. CR-pro-3 In roots of AHA2, the level was significantly reduced by approximately 48% (Fig. 1D). CR-pro-1 , AHA2 CR-pro-5 , AHA2 CR-pro-13 , AHA2 CR-pro-15 The abundance of PM H+-ATPase in leaves of the radish variety was approximately 20%, 37%, 19%, and 19% greater than that of the wild type, respectively (Fig. 1E).

[0098] Furthermore, 4-week-old AHA2 CR-pro PM H+-ATPase hydrolysis activity in mutant leaves was examined by assessing vanadate-sensitive Pi release via ATP hydrolysis. CR-pro-1 , AHA2 CR-pro-5 , AHA2 CR-pro-13 and AHA2 CR-pro-15 PM H+-ATPase activity in leaves of AHA2 significantly increased by 41% to 56% compared to the control. CR-pro-3 and AHA2 CR-pro-12 In leaves of the control group, the amount and activity of PM H+-ATPase were reduced by 39% to 42% (Fig. 1F). CR-pro-1 , AHA2 CR-pro-5 , AHA2 CR-pro-13 The analysis was carried out mainly using lineages.

[0099] Test example 3. AHA2 CR-pro Rhizosphere acidification in the roots of homozygous mutant lines and 15 No. 3 - Accelerated uptake Next, AHA2 CR-pro Root acidification of homozygous mutants was measured using a pH indicator. AHA2 CR-pro-1 , AHA2 CR-pro-5 , and AHA2CR-pro-13 The relative rhizosphere acidification area of ​​the treated soil was approximately 2.6-3.6 times higher than that of the control (Fig. 2B).

[0100] Furthermore, AHA2 CR-pro-1 , AHA2 CR-pro-5 , the nitrate uptake rate in roots of AHA2CR-pro-13, 15 N isotope tracing analysis revealed that AHA2 CR-pro-1 , AHA2 CR-pro-5 In AHA2CR-pro-13, the nitrate uptake rate increased by approximately 13% to 59% compared with the control (Fig. 2C). + When 20 μM vanadate, an inhibitor of α-ATPase, was added to the solution, the rate of nitrate uptake was significantly higher than that of mock (control, AHA2 CR-pro-1 , AHA2 CR-pro-5 , AHA2 CR-pro-13 These results suggest that AHA2 significantly decreased the oxidative stress response compared with the control group (Fig. 2C). CR-pro In the homozygous mutant, the AHA2 level is elevated, resulting in the H + It was suggested that the nitrate uptake rate was significantly improved.

[0101] Test example 4. AHA2 CR-pro Enhanced light-induced stomatal opening and photosynthetic activity in homozygous mutant lines. Increased expression of AHA2 in guard cells promotes light-induced stomatal opening. CR-pro We analyzed the stomatal phenotype and gas exchange parameters of homozygous mutants. CR-pro-1 , AHA2 CR-pro-5 and AHA2 CR-pro-13 The transcription level of AHA2 in guard cells of the stalk was examined by qRT-PCR. CR-pro-1 , AHA2 CR-pro-5 and AHA2 CR-pro-13 The expression of AHA2 in the guard cell-enriched fraction of the stomatal cell-derived stomatal cells was approximately 1.8-2.4 times higher than that in the control (Fig. 3A). + Immunohistochemical detection of AHA2-ATPase protein (Fig. 3B) revealed that AHA2 CR-pro-1 , AHA2 CR-pro-5and AHA2 CR-pro-13 PM H in guard cells of + The amount of α-ATPase protein was approximately 26-31% higher than in the control (Fig. 3C).

[0102] Next, AHA2 CR-pro The light-induced stomatal opening rate was measured using homozygous mutants. CR-pro-1 , AHA2 CR-pro-5 and AHA2 CR-pro-13 The stomatal aperture rate was significantly increased under light conditions compared with the control, and was suppressed by ABA treatment without significant difference (Fig. 3D, E). CR-pro-3 , AHA2 CR-pro-9 and AHA2 CR-pro-12 The stomatal aperture number was significantly reduced in the light condition compared to the control, and a significant difference in the stomatal aperture number was observed between the light and dark conditions (Fig. 3E). CR-pro-1 , AHA2 CR-pro-5 and AHA2 CR-pro-13 The dark-adapted plants were placed under light conditions, and after 50 minutes of light exposure and stabilization, data were collected and calculated. As expected, AHA2 CR-pro-1 , AHA2 CR-pro-5 and AHA2 CR-pro-13 The stomatal conductance and photosynthetic activity of the treated leaves were significantly higher than those of the control leaves (Fig. 3F, G). These results suggest that enhancing AHA2 expression in guard cells by CRISPR / Cas9-mediated promoter editing improves the stomatal aperture rate and photosynthetic capacity of plants.

[0103] Test example 5. AHA2 CR-pro Improved plant growth and seed production in homozygous mutant lines AHA2 CR-pro Phenotypic characteristics of homozygous mutant strains revealed that AHA2 CR-pro-1 , AHA2 CR-pro-5 , AHA2 CR-pro-13 , AHA2 CR-pro-15 and AHA2 CR-pro-16 In AHA2, biomass increased significantly by approximately 21% to 40%. CR-pro-3 , AHA2CR-pro-9 and AHA2 CR-pro-12 In the AHA2 knockout mutant, the cotyledons were reduced by approximately 32% to 38% (Fig. 4A, B). It has been reported that AHA2 knockout mutants have smaller cotyledons. CR-pro The cotyledon diameter of the homozygous mutant lines was also examined. CR-pro-3 , AHA2 CR-pro-9 , the cotyledon diameter of AHA2CR-pro-12 was significantly reduced compared to the control, whereas AHA2 CR-pro-1 , AHA2 CR-pro-5 , AHA2 CR-pro-13 , AHA2 CR-pro-15 , AHA2 CR-pro-16 , AHA2 CR-pro-23 No difference was observed between the two (Fig. 4C). AHA2 is involved in the regulation of root morphology and growth under nutrient deficiency, and we further hypothesized that AHA2 expression under various nutritional stresses, including phosphorus and nitrogen deficiency, may be important. CR-pro The length of the primary root of the homozygous mutant was measured. CR-pro The primary root lengths of the AHA2 and control plants were comparable under normal nutritional conditions (Fig. 4D). CR-pro-3 , AHA2 CR-pro-9 and AHA2 CR-pro-12 The primary root length of AHA2 was reduced by approximately 31-42% under nitrogen deficiency compared to the control, and by 7-20% under phosphorus deficiency (Fig. 4D). CR-pro During the growth period of the mutant and control, AHA2 CR-pro-15 and AHA2 CR-pro-16 The flowering time of the plants was about 4-5 days earlier than that of the control, and the number of rosette leaves was reduced.

[0104] Next, AHA2 CR-pro The agronomic traits and seed production of the mutant lines were investigated over two seasons (summer and autumn) (Fig. 5). CR-pro-1 , AHA2 CR-pro-5 , AHA2 CR-pro-13 , AHA2 CR-pro-15 The number of ovaries in AHA2CR-pro-16 and AHA2CR-pro-16 increased by approximately 15% to 43% in summer and approximately 12% to 33% in autumn compared to the control. CR-pro-3 , AHA2 CR-pro-9and AHA2 CR-pro-12 The AHA2 levels decreased by approximately 9% to 17% in summer and approximately 16% to 25% in autumn (Fig. 5C). CR-pro There was no significant difference in the average weight of each ovary between the homozygous mutant and the control (Fig. 5D). CR-pro-3 Mutant strains and AHA2 CR-pro-12 The AHA2 gene expression level was decreased in the mutant strain, but not in other strains. CR-pro No significant difference was observed in the homozygous mutant strain compared to the control (Fig. 5E). CR-pro-3 and AHA2 CR-pro-12 showed a decrease in seed production of approximately 19% to 25% in summer, followed by a decrease of approximately 23% to 30% in autumn compared to the control. CR-pro-1 , AHA2 CR-pro-5 and AHA2 CR-pro-13 Seed production in the control plants increased by approximately 37% to 53% in summer and 41% to 55% in autumn compared to the control (Fig. 5F). These results clearly demonstrate that enhancing AHA2 transcription levels by multiple promoter editing significantly contributes to plant growth and seed production.

Claims

1. A genome-edited plant body, comprising a mutation of base insertion and / or base deletion in the promoter of the AHA2 gene or its orthologous gene, and the level of the AHA2 protein or its orthologous protein in leaves being improved due to the mutation.

2. The genome-edited plant body according to claim 1, wherein the mutation is present within 4000 b upstream of the start codon of the AHA2 gene or its orthologous gene.

3. The genome-edited plant body according to claim 1, wherein at least one of the mutations is present within 500 b upstream of the start codon of the AHA2 gene or its orthologous gene.

4. The genome-edited plant body according to claim 1, wherein the number of mutation sites is 3 or more.

5. The genome-edited plant body according to claim 1, wherein the number of mutation sites is 3 to 5.

6. The genome-edited plant body according to claim 1, wherein the mutation is present within or near the conserved region of the promoter of the AHA2 gene or its orthologous gene.

7. The genome-edited plant body according to claim 6, wherein the vicinity of the conserved region is within 200 b from the end of the conserved region.

8. The genome-edited plant body according to claim 6, wherein the conserved region is at least one region selected from the group consisting of within 300 b upstream of the start codon of the AHA2 gene or its orthologous gene, 1000 - 1500 b upstream of the start codon of the AHA2 gene or its orthologous gene, 1800 - 2300 b upstream of the start codon of the AHA2 gene or its orthologous gene, and 3000 - 3500 b upstream of the start codon of the AHA2 gene or its orthologous gene.

9. The genome-edited plant body according to any one of claims 1 - 8, wherein the level of the AHA2 protein or its orthologous protein is improved by 1.1 times or more.

10. The genome-edited plant body according to any one of claims 1 - 8, which is a Brassicaceae plant.

11. Seeds produced from the genome-edited plant body according to any one of claims 1 - 8.

12. A method for producing a genome-edited plant body according to any one of claims 1 - 8, comprising the steps of performing genome editing to introduce a mutation of base insertion and / or base deletion in the promoter of the AHA2 gene or its orthologous gene of a plant, and selecting a plant body using the level of the AHA2 protein or its orthologous protein in leaves as an indicator.

13. A method for screening a plant body having improved rhizosphere acidification ability and / or nitrogen absorption ability by roots and improved growth ability, comprising the steps of performing genome editing to introduce a mutation of base insertion and / or base deletion into the promoter of the AHA2 gene of the plant or its orthologous gene, and selecting the plant body using the level of the AHA2 protein or its orthologous protein in the leaf as an index.

Images