Stress-resistant plant

Abstract

The present invention relates to, inter alia, a gene involved in the reactive oxygen species (ROS) resistance of a plant, a plant cell having ROS resistance utilizing the gene, and a plant that has increased CO2 fixation and that is configured from the plant cell. More specifically, a plant cell according to the present invention has reactive oxygen species (ROS) resistance and includes a modification of at least one endogenous gene according to any of the following (a)-(c) that reduces the function of the gene: (a) a gene including a base sequence represented by any of SEQ ID NOs: 1-8; (b) a gene including a base sequence having 80% or greater sequence identity with a base sequence represented by any of SEQ ID NOs: 1-8; and (c) a gene that is capable of hybridizing, under stringent conditions, with a gene including a base sequence represented by any of SEQ ID NOs: 1-8.

Description

Stress-tolerant plants

[0001] Related Applications: This specification includes the contents of the specification of Japanese Patent Application No. 2021-56055 (filed March 29, 2021), which is the priority basis of this application. Technical Field: The present invention relates to a gene involved in plant reactive oxygen species (ROS) resistance, a plant cell having ROS resistance using said gene, and a CO2-producing plant cell comprising said plant cell. ２ This relates to plants with increased fixed amounts.

[0002] Since the Industrial Revolution, CO has been emitted from human activities such as the burning of fossil fuels. ２ Atmospheric CO due to emissions ２ The concentration is increasing, and CO ２ Reducing CO emissions is a global issue. ２ Fixation of CO ２ By increasing the amount of CO2 fixed, ２ This may prevent the concentration from increasing.

[0003] CO by plants ２ It is thought that improving photosynthetic efficiency is necessary to increase fixation efficiency, but plants are constantly exposed to external stresses (strong light, low temperature, high temperature, drought, salt, air pollution, pathogens, etc.), and it is known that these environmental stresses reduce photosynthetic efficiency via reactive oxygen species (ROS) (Non-Patent Documents 1 and 2).

[0004] Therefore, it is expected that a decline in photosynthetic efficiency can be prevented by increasing the ROS resistance or ROS removal ability of plants, and that the stress resistance of plants can be improved by expressing specific genes or by applying specific substances to plants, thereby increasing biomass productivity (CO ２ Research is currently underway to improve the fixed amount (Non-Patent Documents 3 and 4).

[0005] Most of the carbon fixation by photosynthesis on earth is caused by trees (forests), and CO ２ If the fixation efficiency can be improved, CO ２ It is believed that this will make a significant contribution to problem solving.

[0006] Choudhury et al., Plant Signaling & Behavior, 8:4, e23681, 2013Frontiers in Plant Science, Vol 7, article 187, 2016, doi: 10.3389 / fpls.2016.00187Kudo et al., The Plant Journal, Vol 97, 240-256, 2019Yoshida et al. al., Applied Microbiology and Biotechnology 60, 665-670, 2003

[0007] The object of the present invention is to improve the ROS tolerance of plants and ２ The object of the present invention is to provide a new means for improving fixing efficiency.

[0008] The inventors conducted research on Japanese cedar, the most abundant forest resource in Japan, focusing on genes whose disruption confers ROS resistance, and verified the effects. They identified genes involved in ROS resistance in plants and confirmed that disrupting these genes improves ROS resistance in plants.

[0009] The present invention is based on the above research results and relates to the following [1] to

[12] . [1] A plant cell having reactive oxygen species (ROS) tolerance, the plant cell comprising a modification that reduces the function of at least one of the following endogenous genes (a) to (c): (a) a gene comprising a nucleotide sequence set forth in any of SEQ ID NOs: 1 to 8; (b) a gene comprising a nucleotide sequence having 80% or more sequence identity with a nucleotide sequence set forth in any of SEQ ID NOs: 1 to 8, the reduced function of which confers ROS tolerance in the plant; or (c) a gene that hybridizes under stringent conditions with a gene comprising a nucleotide sequence set forth in any of SEQ ID NOs: 1 to 8, the reduced function of which confers ROS tolerance in the plant. [2] The plant cell according to [1], wherein the modification that reduces the function of the endogenous gene is a mutation in the genomic sequence that results in the loss of function of the endogenous gene. [3] The plant cell according to [1], wherein the modification that reduces the function of the endogenous gene is a mutation that reduces or disrupts expression of the endogenous gene. [4] The plant cell according to [1], wherein the modification that reduces the function of the endogenous gene is one or more indels in the ORF region of the endogenous gene. [5] A plant or a part thereof constituted by the plant cell according to any one of claims [1] to [4]. [6] The plant or a part thereof according to [5], wherein the part of the plant is any one selected from the group consisting of callus, plant tissue, plant tissue culture, seeds, fruits, leaves, stems, trunks, roots, and flowers. [7] CO of the plant ２[8] A plant or part thereof according to [5] or [6], wherein the amount of fixation is increased. [8] Any of the following genes (a) to (c): (a) a gene comprising a nucleotide sequence shown in any of SEQ ID NOs: 1, 3, 5, 7, and 8; (b) a gene comprising a nucleotide sequence having 80% or more sequence identity with the nucleotide sequence shown in any of SEQ ID NOs: 1, 3, 5, 7, and 8, the loss of which function confers reactive oxygen species (ROS) resistance in a plant; (c) a gene that hybridizes under stringent conditions with a gene comprising a nucleotide sequence shown in any of SEQ ID NOs: 1, 3, 5, 7, and 8, the loss of which function confers ROS resistance in a plant. [9] The plant cell according to any of [1] to [4], the plant or part thereof according to any of [5] to [7], or the gene according to [8], wherein the plant is a plant selected from the Cupressaceae family, Pinaceae family, Myrtaceae family, and Betulaceae family.

[10] CO ２ A method for producing a plant with an increased amount of CO immobilization, the method comprising obtaining plant cells containing a modification that reduces the function of at least one reactive oxygen species (ROS) resistance-related gene, and generating a plant from the plant cells, wherein the ROS resistance-related gene is any of the following (a) to (c): (a) a gene containing a nucleotide sequence set forth in any of SEQ ID NOs: 1 to 8, (b) a gene containing a nucleotide sequence that has 80% or more sequence identity with the nucleotide sequence set forth in any of SEQ ID NOs: 1 to 8, the reduced function of which confers ROS resistance in the plant, or (c) a gene that hybridizes under stringent conditions with a gene containing a nucleotide sequence set forth in any of SEQ ID NOs: 1 to 8, the reduced function of which confers ROS resistance in the plant.

[11] ２A method for increasing the amount of immobilization, comprising modifying at least one ROS resistance-associated gene to reduce its function, and generating a plant from the plant cell, wherein the ROS resistance-associated gene is any of the following (a) to (c): (a) a gene comprising a nucleotide sequence set forth in any of SEQ ID NOs: 1 to 8, (b) a gene comprising a nucleotide sequence having 80% or more sequence identity with a nucleotide sequence set forth in any of SEQ ID NOs: 1 to 8, the reduced function of which confers ROS resistance in the plant, or (c) a gene that hybridizes under stringent conditions with a gene comprising a nucleotide sequence set forth in any of SEQ ID NOs: 1 to 8, the reduced function of which confers ROS resistance in the plant.

[12] The method according to

[10] or

[11] , wherein the plant is any plant selected from the Cupressaceae family, Pinaceae family, Myrtaceae family, and Betulaceae family.

[0010] According to the present invention, the ROS tolerance of plants is increased, thereby increasing the CO ２ In particular, the amount of CO2 fixed can be increased for cedar, which is the most abundant forest resource in Japan. ２ Increasing the fixed amount is useful for environmental problems such as global warming.

[0011] Unlike conventional mutation breeding, which requires growing plants to make judgments, the method of the present invention allows for the screening and evaluation of ROS resistance-related genes in a short period of time by modifying the genome of cultured cells (callus).

[0012] Figure 1 shows the structure of the genome editing vector (pGECj). Figure 2 shows the results of confirming sequence mutations in the #SR10 strain. M: 100bp Ladder Marker. Lane 1: #SR10 strain (SR10 gene target region PCR product) (untreated). Lane 2: parent strain (SR10 gene target region PCR product) (untreated). Lane 3: #SR10 strain (SR10 gene target region PCR product) (restriction enzyme (TaqI) treatment). Lane 4: parent strain (SR10 gene target region PCR product) (restriction enzyme (TaqI) treatment). Figure 3 shows the results of confirming sequence mutations in the #SR11 strain. M: 100 bp Ladder Marker; Lane 1: #SR11 strain (SR11 gene target region PCR product) "untreated with restriction enzyme"; Lane 2: parent strain (SR11 gene target region PCR product) "untreated with restriction enzyme"; Lane 3: #SR11 strain (SR11 gene target region PCR product) "treated with restriction enzyme (HaeIII)"; Lane 4: parent strain (SR11 gene target region PCR product) "treated with restriction enzyme (HaeIII)" Figure 4 shows the results of confirming sequence mutations in #SR13 strain. M: 100-bp Ladder Marker; Lane 1: #SR13 strain (SR13 gene target region PCR product) "untreated with restriction enzyme"; Lane 2: parent strain (SR13 gene target region PCR product) "untreated with restriction enzyme"; Lane 3: #SR13 strain (SR13 gene target region PCR product) "treated with restriction enzyme (Hpy188I)"; Lane 4: parent strain (SR13 gene target region PCR product) "treated with restriction enzyme (Hpy188I)" Figure 5 shows the results of confirming sequence mutations in #SR14 strain. M: 100-bp Ladder Marker; Lane 1: #SR14 strain (SR14 gene target region PCR product) "untreated with restriction enzyme"; Lane 2: parent strain (SR14 gene target region PCR product) "untreated with restriction enzyme"; Lane 3: #SR14 strain (SR14 gene target region PCR product) "treated with restriction enzyme (Hpy188I)"; Lane 4: parent strain (SR14 gene target region PCR product) "treated with restriction enzyme (Hpy188I)" Figure 6 shows the results of confirming sequence mutations in #SR15 strain.M: 100-bp Ladder Marker; Lane 1: #SR15 strain (SR15 gene target region PCR product) "untreated with restriction enzyme"; Lane 2: parent strain (SR15 gene target region PCR product) "untreated with restriction enzyme"; Lane 3: #SR15 strain (SR15 gene target region PCR product) "treated with restriction enzyme (NlaIII)"; Lane 4: parent strain (SR15 gene target region PCR product) "treated with restriction enzyme (NlaIII)"; Figure 7 shows the results of confirming sequence mutations in #SR17 strain. M: 100-bp Ladder Marker; Lane 1: #SR17 strain (SR17 gene target region PCR product) "untreated with restriction enzyme"; Lane 2: parent strain (SR17 gene target region PCR product) "untreated with restriction enzyme"; Lane 3: #SR17 strain (SR17 gene target region PCR product) "treated with restriction enzyme (HaeIII)"; Lane 4: parent strain (SR17 gene target region PCR product) "treated with restriction enzyme (HaeIII)"; Figure 8 shows the results of confirming sequence mutations in #SR24 strain. M: 100-bp Ladder Marker; Lane 1: #SR24 strain (SR24 gene target region PCR product) "untreated with restriction enzyme"; Lane 2: parent strain (SR24 gene target region PCR product) "untreated with restriction enzyme"; Lane 3: #SR24 strain (SR24 gene target region PCR product) "treated with restriction enzyme (AluI)"; Lane 4: parent strain (SR24 gene target region PCR product) "treated with restriction enzyme (AluI)" Figure 9 shows the results of confirming sequence mutations in #SR25 strain. M: 100 bp Ladder Marker, Lane 1: #SR25 strain (SR25 gene target region PCR product) "untreated with restriction enzyme", Lane 2: parent strain (SR25 gene target region PCR product) "untreated with restriction enzyme", Lane 3: #SR25 strain (SR25 gene target region PCR product) "treated with restriction enzyme (TaqI)", Lane 4: parent strain (SR25 gene target region PCR product) "treated with restriction enzyme (TaqI)". Figure 10 shows the results of growth responses in various media.A: Control, B: 2mM menadione, C: 30µM menadione, D: 1µM paraquat; the arrangement of each clone during growth evaluation is shown on the right of Figure 10 (top row from left: #SR10, #SR11, #SR13, #SR14; middle row from left: #SR15, #SR17, #SR24, #SR25; bottom row center: parent plant). Figure 11 shows regenerated individuals obtained by regeneration treatment of genome-edited callus and parent plant callus. Germination and root formation were confirmed for all genome-edited individuals, and the germination and rooting rates were comparable to those of the parent plant.

[0013] 1. Plant Cells with Reactive Oxygen Species (ROS) Tolerance The present invention relates to plant cells with reactive oxygen species (ROS) tolerance. The plant cells of the present invention are characterized in that they contain a modification that reduces the function of at least one of the following endogenous genes (a) to (c): (a) a gene comprising a nucleotide sequence set forth in any of SEQ ID NOs: 1 to 8; (b) a gene comprising a nucleotide sequence that has 80% or more sequence identity with a nucleotide sequence set forth in any of SEQ ID NOs: 1 to 8, the reduced function of which confers ROS tolerance to the plant; or (c) a gene that hybridizes under stringent conditions with a gene comprising a nucleotide sequence set forth in any of SEQ ID NOs: 1 to 8, the reduced function of which confers ROS tolerance to the plant.

[0014] As used herein, "reactive oxygen species (ROS)" refers to oxygen molecules (O ２ ) is a general term for a group of highly reactive molecules derived from hydrogen peroxide (H ２ O ２ ), superoxide anion radical (O ２ － ), hydroxyl radical (OH ・ ), singlet oxygen, etc. In plant cells, many environmental stresses generate ROS, which affect plant growth and productivity.

[0015] (a) a gene comprising a nucleotide sequence shown in any one of SEQ ID NOs: 1 to 8; (b) a gene comprising a nucleotide sequence having 80% or more sequence identity with the nucleotide sequence shown in any one of SEQ ID NOs: 1 to 8, the loss of which function confers ROS resistance in plants; (c) a gene that hybridizes under stringent conditions with a gene comprising a nucleotide sequence shown in any one of SEQ ID NOs: 1 to 8, the loss of which function confers ROS resistance in plants.

[0016] As used herein, the term "plant cells" is not particularly limited, but is preferably a cell of a woody plant (tree), and more preferably a cell of a plant of the Cupressaceae, Pinaceae, Myrtaceae, or Betulaceae families.

[0017] Examples of plants of the Cupressaceae family include the following plants (Gadek et al., 2000): Among these, plants of the Cupressaceae subfamily are preferred, with cedar being more preferred. Subfamily Cupressaceae: genus Chamaecyparis obtusa (Chamaecyparis obtusa, etc.), genus Thujopsis dolabrata (Thujopsis dolabrata, etc.), genus Juniperus (Juniperus japonicus, etc.), genus Thujopsis occidentalis (Thujopsis occidentalis, etc.), genus Cypress (Cypress cypress, etc.), genus Oriental cypress (Oriental cypress, etc.), genus Thujopsis arvensis (Thujopsis arvensis, etc.), genus Thujopsis rhomboidea (Thujopsis rhomboidea, etc.); Subfamily Callitrinae: genus Actinostrobus (A. acuminatus), genus Austrocedrus (A. chiliensis), genus Callitris (C. rhomboidea), genus Diselma (D. archeri), genus Fitzroya (F. aupressoides), genus Libocedrus (L. bidwillii), genus Neocallitropsis (N. araucarioides), Papuacedrus (P. papuana), Pilgerodendron (P. uviferum), Widdringtonia (W. schwartzii); Crypoideae: Cryptomeria japonica (Japanese cedar), Bald cypress (bald cypress, etc.), Celastrus serrata (Crystals of the Cedar family); Sequoiae: Sequoia (Sequoia), Sequoiadendron (Sequoiadendron), Metasequoia (Metasequoia); Crypoideae: Celastrus japonica (Crystals of the Cedar family, etc.); Crypoideae: Celastrus japonica (Crystals of the Cedar family, etc.); Crypoideae: Celastrus japonica (Crystals of the Cedar family, etc.).

[0018] Examples of plants of the Pinaceae family include the following plants: Among these, plants of the subfamily Pinaceae and subfamily Picea are preferred, with red pine, Scotch pine and Norway spruce being more preferred. Subfamily Pinaceae: genus Pinus (Swiss pine, lodgepole pine, Japanese red pine, Tago-sho, Ryukyu pine, longleaf pine, Japanese white pine, northern red pine, Japanese stone pine, radiata pine, Pinus rigida, Scotch pine (also known as Scots pine), loblolly pine, Japanese black pine, etc.); Subfamily Picea: genus Picea (European spruce (also known as Norway spruce, Norway spruce, European spruce), Engelmann spruce, Picea glehnii, Picea japonicus, etc.); Subfamily Larix: genus Larix (European larch, Picea dactylon, Larch, Japanese larch, etc.), Picea spp. (Douglas fir, etc.); Abies subfamily: Abies (fir, giant fir, white fir, mountain balsam fir, mariesii, Nordmann's fir, Abies sachalinensis, white fir, etc.), Cedrus, Cedar, Tsuga, Larix, Tsuga, Tsuga (Japanese hemlock, Tsuga diversifolia, etc.).

[0019] Examples of plants of the Myrtaceae family include the following plants. Among them, plants of the Leptospermoideae subfamily are preferred, with Eucalyptus being more preferred. Leptospermoideae subfamily: Callistemon (Callistemon, etc.), Eucalyptus (Eucalyptus, etc.), Leptospermum (Leptospermum, etc.), Callistemon (Tea tree, etc.); Myrtoideae subfamily: Eugenia (Pitanga, etc.), Ogasawara (Eugenia, Pohutukawa, Hawaiian Myrtus, Rata, Southern Rata, etc.), Rubus (Camu Camu, etc.), Myrtle (Myrtle, etc.), Pimenta (Allspice, etc.), Psammophila (Guava, etc.), Myrtaceae (Myrtaceae, etc.), Myrtaceae (Myrtaceae, Clove, Adiantum, Myrtaceae, Waxberry, etc.).

[0020] Examples of plants of the Betulaceae family include the following plants. Among them, the subfamily Betulaceae is preferred, and white birch is more preferred. Subfamily Betulaceae: Alder, Betulaceae (white birch, etc.); Subfamily Hazelinae: Carpinus (carpinus, carpinus, red carpinus, etc.), Corylus (hazel, hornbeam, corylus avellana, etc.), and Lithospermum (lily of the valley, etc.).

[0021] The plant cells of the present invention contain a modification that reduces the function of at least one "ROS resistance-associated gene" as described in detail in a later chapter of this specification.

[0022] Here, "decreased function" refers to the inhibition, reduction, or loss of the biological function of a ROS resistance-associated gene, including, for example, the inhibition, reduction, or loss of the biological function of a ROS resistance-associated gene due to the inhibition, reduction, or loss of the expression of the gene, the inhibition, reduction, or loss of the expression of a protein that is the translation product of the ROS resistance-associated gene, and the inhibition, reduction, or loss of the biological function of the gene caused by a structural change or functional suppression of the protein.

[0023] The "modification" that reduces the function of a ROS resistance-associated gene is an artificial modification and does not include natural mutations. The "modification" may be a modification (mutation, modification) of the ROS resistance-associated gene itself, a modification (mutation, modification) of a region involved in transcriptional control of the ROS resistance-associated gene, or the introduction of a foreign gene or foreign substance.

[0024] Preferably, the "modification" is a mutation in the genomic sequence that results in a reduction or loss of function of the ROS resistance-associated gene, more preferably a loss of function, and even more preferably a mutation that disrupts expression of the ROS resistance-associated gene. Examples of mutations that disrupt expression of the ROS resistance-associated gene include one or more indels in the ORF region of the ROS resistance-associated gene.

[0025] Methods for introducing the "modification" into plant cells include, for example, genome editing using site-specific nucleases or deaminases, oligonucleotide-directed mutagenesis, RNA interference, the use of antisense or ribozymes, etc., but are not limited to these methods as long as they do not deviate from the spirit of the present invention. From the standpoint of breeding efficiency and modification accuracy, genome editing using site-specific nucleases is preferred.

[0026] [Genome Editing] a) Site-specific Nucleases Site-specific nucleases can be used to introduce mutations into ROS resistance-related genes that alter their expression levels or structure. Examples of site-specific nucleases include zinc finger nucleases (ZFNs), transactivator-like effector nucleases (TALENs), and Cas9, Cas3, Cas12a, and Cas nickase (Cpf1) from the CRISPR / Cas system.

[0027] b) PPR (pentatricopeptide repeat) PPR (pentatricopeptide repeat) is a protein found in plant cells that consists of a repeating structure of 35 amino acids. It is a nucleic acid-binding protein that specifically binds to RNA base sequences. This PPR can be used to specifically cleave RNA or to replace specific bases.

[0028] c) Oligonucleotide Directed Mutagenesis (ODN) technology: An oligonucleotide or a short single-stranded DNA fragment (approximately 20 to 30 bases long) that is homologous to a target base sequence on the genome and has a mutation of about one base is synthesized, and then directly introduced into a cell using a particle gun method or the like, thereby introducing a mutation into the sequence of a specific gene.

[0029] d) Nucleotide rewriting: This method uses deaminase to rewrite nucleotides, thereby inducing point mutations. When combined with the CRISPR system, genome editing can also be performed by introducing site-specific point mutations.

[0030] [Gene Recombination] a) RNAi Method By introducing and expressing 21-23 bp short double-stranded RNA (siRNA: small interfering RNA) or long double-stranded RNA (dsRNA: double-strand RNA), gene expression can be suppressed by cleaving the homologous portion of the transcription product (mRNA) of the target gene.

[0031] b) Antisense Method By expressing RNA complementary to the mRNA of a target gene, double-stranded RNA is produced, which can inhibit transcription of mRNA or degrade mRNA, thereby suppressing gene expression.

[0032] c) Use of Ribozymes By introducing and expressing catalytic RNA (ribozymes), it is possible to cleave transcribed RNA and control the expression of target genes.

[0033] d) Gene targeting (homologous recombination) This is a technique for modifying endogenous genes using homologous recombination. Although there have been few successful cases in plants, it is possible to replace the target gene region with a mutated gene or to delete it.

[0034] e) Introduction of Dysfunctional Genes: This method involves introducing a gene that has been mutated to eliminate its function, and then expressing it in large quantities, thereby suppressing the activity of functional genes. For example, if a regulatory gene binds to DNA, the function of the regulatory gene can be suppressed by occupying that region with a nonfunctional gene (because it is expressed in large amounts).

[0035] [Others] a) RNA-dependent DNA methylation: This is a technology that controls the expression of a gene by methylating some bases in a region involved in the gene's expression. When a DNA fragment homologous to a partial sequence (target sequence) in the promoter region of a gene whose expression is to be suppressed is introduced into a cell, some bases in the target sequence are methylated via mRNA, the transcription product of the DNA, thereby suppressing (inactivating) the expression of the gene. Even after the DNA fragment disappears, the methylation state of the promoter is inherited for at least several generations. Therefore, if the RNA production gene is not integrated into the genome of the target organism or is removed after integration, the expression of a specific endogenous gene can be suppressed without changing the base sequence information on the organism's original genome.

[0036] b) Indels in Promoter Regions By introducing DNA insertions or deletions into the promoter region of a target gene, its transcription function can be suppressed or eliminated, thereby suppressing the expression of a specific endogenous gene.

[0037] The plant cells of the present invention have higher ROS resistance than wild-type cells due to the above-mentioned modifications that result in reduced function of ROS resistance-related genes.

[0038] 2. ROS-Resistant Plants or Parts Thereof The present invention also provides plants or parts thereof that are composed of the above-mentioned plant cells. As used herein, the term "plant part" is not particularly limited and may include callus, plant tissue, plant tissue culture, seeds, fruits, leaves, stems, trunks, roots, and flowers.

[0039] The plants of the present invention have higher ROS resistance than wild-type plants. Imparting ROS resistance confers resistance to environmental stress to the plants, alleviating the reduction in photosynthetic efficiency, and as a result, CO ２ Increase the amount of fixation.

[0040] 3. ROS Resistance-Associated Gene The "ROS resistance-associated gene" of the present invention is a gene identified as an endogenous gene whose reduced function confers ROS resistance to a plant.

[0041] 3.1 Identification of ROS Resistance-Associated Genes The "ROS resistance-associated genes" of the present invention can be screened and identified, for example, by the following two-stage test.

[0042] [First test: extraction of candidate genes] 1) A step of mutagenizing plant cells to randomly introduce mutations to prepare a mutant cell library; 2) A step of selecting cell lines having ROS resistance from the mutant cell library; and 3) A step of comparing the sequences of the selected cell lines with those of wild-type strains to extract endogenous genes whose expression is significantly suppressed compared to the wild-type strain, or endogenous genes having mutations in the selected cell lines.

[0043] In step 1, the mutation library can be constructed according to methods known in the art. The method for introducing mutations is not particularly limited, and the plant cells can be subjected to mutation treatments such as treatment with alkylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine and ethylmethanesulfonate, treatment with nucleic acid base analogs such as 5-bromouracil and 2-aminopurine, irradiation with energy rays such as gamma rays, X-rays, and heavy ion beams, or genetic manipulation using transposons or T-DNA. Examples of plant cells that can be used include those described in "1. Plant Cells Resistant to Reactive Oxygen Species (ROS)." For example, the callus of Cryptomeria japonica that is capable of somatic embryogenesis, as described in the Examples, can be used.

[0044] In step 2, ROS-resistant cell lines can be selected by culturing the mutant cell library in a selective medium (applying ROS stress) and selecting ROS-resistant mutants from surviving colonies. The selective medium is not particularly limited as long as it is suitable for the growth of the plant cells used, but a solid medium is preferred. Methods for applying ROS stress include adding a direct ROS source such as hydrogen peroxide, adding an ROS generator such as paraquat, or adding a drug that inhibits the ROS removal metabolism of cells, such as hydroxyurea or aminooxyacetic acid. ROS-resistant strains are selected from those with improved ROS resistance compared to the parent strain. If necessary, cells may be exposed to multiple different stress conditions to evaluate the degree of ROS resistance, and strains that exhibit stronger ROS resistance under various ROS stress environments may be selected.

[0045] In step 3, candidate genes can be extracted from the ROS-resistant strains using, for example, RNA-Seq analysis. The analysis is preferably performed by combining expression analysis (extraction of genes whose expression levels are suppressed compared to the parent strain) and mutation analysis (extraction of genes that are likely to have lost their function due to mutation).

[0046] [Second test: functional evaluation] 1) A step of producing a plant cell into which a modification that reduces the function of any one of the endogenous genes extracted in the first test is introduced, by targeting the gene; and 2) A step of evaluating the ROS resistance of the plant cell and identifying the endogenous gene whose modification confers ROS resistance as a ROS resistance-related gene.

[0047] In step 1, the "modification" that reduces the function of the endogenous gene may be any of the "modifications" described in "1. Plant cells with reactive oxygen species (ROS) resistance." Preferably, the "modification" is a mutation in the genomic sequence that results in loss of function of the endogenous gene, more preferably a mutation that disrupts expression of the endogenous gene, such as one or more indels in the ORF region of the endogenous gene.

[0048] The method for introducing the "modification" into plant cells can also be the method described in "1. Plant cells resistant to reactive oxygen species (ROS)." Examples include genome editing using site-specific nucleases or deaminases, oligonucleotide-directed mutagenesis, RNA interference, and the use of antisense or ribozymes. Among these, genome editing using site-specific nucleases, particularly the introduction of indels in the ORF region of the target gene, is preferred from the standpoint of simplicity and efficiency.

[0049] 3.2 Nucleotide Sequences of ROS Resistance-Associated Genes Suitable examples of the "ROS resistance-associated genes" of the present invention include genes comprising the nucleotide sequences (cDNA sequences) set forth in any of SEQ ID NOS: 1 to 8. The "ROS resistance-associated genes" of the present invention are not limited to the aforementioned sequences, and genes having nucleotide sequences with high sequence identity to the aforementioned nucleotide sequences are also encompassed in the "ROS resistance-associated genes" of the present invention, as long as a decrease in their function confers ROS resistance to plants.

[0050] For example, a gene that has 50% or more, 60% or more, 70% or more, preferably 80% or more, more preferably 90% or more, and even more preferably 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the base sequence set forth in any of SEQ ID NOs: 1 to 8, and whose functional impairment confers ROS resistance to a plant, is also encompassed by the "ROS resistance-associated gene" of the present invention.

[0051] Genes that can hybridize under stringent conditions to a gene containing (preferably consisting of) a nucleotide sequence shown in any one of SEQ ID NOs: 1 to 8, and genes whose loss of function confers ROS resistance to plants, are also encompassed in the "ROS resistance-associated genes" of the present invention.

[0052] Stringent conditions include, but are not limited to, hybridization conditions in which a nylon membrane with immobilized DNA is incubated with a probe for 20 hours at 65°C in a solution containing 6xSSC (1xSSC is 8.76g sodium chloride, 4.41g sodium citrate dissolved in 1L of water), 1% SDS, 100μg / ml salmon sperm DNA, 0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone, and 0.1% Ficoll. Those skilled in the art can set hybridization conditions by taking into account conditions such as the salt concentration of the buffer, temperature, and other conditions such as probe concentration, probe length, and reaction time. Post-hybridization washing conditions include, for example, "2xSSC, 0.1% SDS, 42°C" and "1xSSC, 0.1% SDS, 37°C." More stringent conditions include, for example, "1xSSC, 0.1% SDS, 65°C" and "0.5xSSC, 0.1% SDS, 50°C."

[0053] The "ROS resistance-associated gene" is preferably a gene derived from a plant of the Cupressaceae, Pinaceae, Myrtaceae, or Betulaceae family.

[0054] The deduced ORFs of the "ROS resistance-associated genes" of the present invention are set forth in SEQ ID NOs: 9 to 16, and encode the deduced amino acid sequences set forth in SEQ ID NOs: 17 to 24, respectively. The "ROS resistance-associated genes" of the present invention also include genes that encode amino acid sequences highly homologous to SEQ ID NOs: 17 to 24, as long as a decrease in their function confers ROS resistance to plants.

[0055] For example, a gene that encodes an amino acid sequence that has 80% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 96% or more, 97% or more, 98% or more, or 99% or more homology to any of the amino acid sequences set forth in SEQ ID NOs: 17 to 24, and whose functional impairment confers ROS resistance to a plant, is also encompassed by the "ROS resistance-associated gene" of the present invention.

[0056] Portions of the cDNA sequences of the ROS resistance-associated genes shown in SEQ ID NOs: 1 to 8, the base sequences of the putative ORFs of the ROS resistance-associated genes shown in SEQ ID NOs: 9 to 16, and the RNA sequences corresponding to the sequences can be used as primers for amplifying the ROS resistance-associated genes or as guide RNA sequences for genome editing.

[0057] For example, RNAs containing 17 to 25 consecutive bases in any of the base sequences shown in SEQ ID NOs: 1 to 8 or 9 to 16, or base sequences complementary to the sequences, can be used as guide RNAs for genome editing of ROS resistance-related genes (provided that T in the SEQ ID NOs: 1 to 8 is replaced with U). Examples of such guide RNAs are shown in SEQ ID NOs: 25 to 32 (provided that T in the SEQ ID NOs: 25 to 32 is replaced with U).

[0058] Furthermore, DNA containing 17 to 25 consecutive bases in any of the base sequences shown in SEQ ID NOS: 1 to 8 or 9 to 16, or a base sequence complementary to any of the above sequences, can be used as a primer for specifically amplifying ROS resistance-associated genes. Examples of such primers are shown in SEQ ID NOS: 33 to 48.

[0059] 4. CO in plants ２As mentioned above, many environmental stresses are factors that reduce the photosynthetic efficiency of plants through ROS. Improving the ROS tolerance of plants will prevent the reduction in photosynthetic efficiency due to environmental stress, thereby increasing the amount of CO fixation. ２ The present invention provides a method for increasing the amount of CO fixation in plants by utilizing the ROS resistance-related gene. ２ Methods for increasing the amount of fixation and CO ２ A method for producing plants with increased fixation levels is provided.

[0060] The CO of the plant according to the present invention ２ The method for increasing the immobilized amount comprises modifying at least one ROS resistance-related gene to reduce its function, and generating a plant from the plant cell.

[0061] According to the present invention, CO ２ The method for producing a plant with an increased immobilization amount is characterized by obtaining a plant cell containing a modification that reduces the function of at least one ROS resistance-related gene, and generating a plant body from the plant cell.

[0062] The ROS resistance-associated gene to be used is not particularly limited as long as it is identified by the method specified in Section 3.1 above. Suitable examples of ROS resistance-associated genes are the genes described in Section 3.2 above, and include, for example, the following genes (a) or (b): (a) a gene comprising a nucleotide sequence set forth in any of SEQ ID NOs: 1 to 8, (b) a gene comprising a nucleotide sequence having 80% or more sequence identity with a nucleotide sequence set forth in any of SEQ ID NOs: 1 to 8, the loss of which function confers reactive oxygen species (ROS) resistance in plants.

[0063] The "modification that reduces function" and the method of "modification" are as described in "1. Plant cells having resistance to reactive oxygen species (ROS)."

[0064] CO produced by the method of the present invention ２Plants with increased fixation levels are useful for addressing environmental issues such as global warming. Furthermore, the method of the present invention, which involves screening and evaluating ROS resistance-related genes through genome modification of cultured cells (callus), allows for the breeding of desired ROS-resistant plants in a short period of time, unlike conventional mutation breeding, which involves growing plants and then making evaluations.

[0065] The present invention will be specifically explained below using Reference Examples and Examples, but the present invention is not limited to these Examples.

[0066] Reference Example 1: Identification of ROS resistance-related genes 1. Extraction of candidate genes by RNA-seq analysis ROS-resistant mutant strains were induced, and for mutant strains with particularly high ROS resistance, resistance-related gene candidates were extracted by RNA-seq analysis.

[0067] We performed mutagenesis on cultured cells (callus) of Japanese cedar (Cryptomeria japonica) to obtain a mutant cell library with randomly induced mutations. The library was then plated on selective medium and cultured under ROS stress, allowing us to select ROS-resistant mutants from the library.

[0068] By comparing the sequences of the ROS-resistant strain and the parent strain using RNA-seq, we extracted transcription factors that negatively regulate ROS stress or whose expression was significantly suppressed compared to the parent strain (61 genes extracted). Furthermore, we identified the mutation sites in the ROS-resistant mutant strains and extracted genes with mutations that were likely to result in loss of function from the confirmed mutations (21 genes extracted). From the extracted genes, we selected 31 genes that were deemed most likely to be functional loss and set them as targets for the next stage.

[0069] 2. Identification of ROS resistance-related genes by knockout: By knocking out 31 genes extracted by RNA-seq analysis using genome editing, we identified genes (SR10, SR11, SR13, SR14, SR15, SR17, SR24, and SR25) that improve the ROS stress resistance of Cryptomeria japonica callus. The sequence information for the eight identified genes is shown in the table below and in the sequence table.

[0070]

[0071] Of the eight genes, four genes, SR11, SR13, SR14, and SR17, were found to have 90.7-100% identical nucleotide sequences to known genes. However, no information on gene knockout / knockdown or reports indicating a relationship between these genes and ROS resistance were found. (The nucleotide sequence of SR11 shares 99.7% identity with the gene registered at GenBank AK408222.1. The nucleotide sequence of SR13 shares 90.7% identity with the gene registered at GenBank: KY010969.1. The nucleotide sequence of SR14 shares 100% identity with the gene registered at GenBank: AK414819.1. The nucleotide sequence of SR17 shares 100% identity with the genes registered at GenBank: AK413080.1 and GenBank: AK416259.1.) For the other four genes (SR10, SR15, SR24, and SR25), no genes with similar nucleotide sequences (identity of 80% or more) were found.

[0072] Example 1: Preparation of ROS-resistant plants 1. Cultivation of Sugi callus (somatic embryogenic cells) In this example, callus capable of somatic embryogenesis of Cryptomeria japonica (hereinafter referred to as Sugi callus) was used.

[0073] Sugi callus was cultured in 1 / 2MD medium, which contained the following composition: 1.315 g / L Murashige & Skoog Medium Mod. No. 3B [Duchefa Biochemie], 30 g / L sucrose, 2 μM 2,4-dichlorophenoxyacetic acid, 0.5 g / L glutamine, and 1 g / L casein hydrolysate [Casamino Acid, Vitamin Assay / Difco]. The medium pH was adjusted to 5.6–5.8, and solid medium was supplemented with 4 g / L Gelrite or 10 g / L agar. Culture was performed aseptically at 25°C. Sugi callus was subcultured on solid medium every 1–2 weeks.

[0074] 2. Disruption of target genes using genome editing (CRISPR / Cas9 system) - Overview of target gene disruption method using genome editing The CRISPR / Cas9 system was used to disrupt target genes. Specifically, a vector expressing SpCas9 derived from Streptococcus pyogenes and a guide RNA (gRNA) containing a 20-base sequence (guide sequence) complementary to a specific site in the target gene was introduced into Japanese cedar callus, and repair errors (base deletions, insertions, etc.) that occur during the DNA repair process after double-stranded DNA cleavage at the target site by the Cas9 protein were induced.

[0075] Guide sequence design: The 20 bases upstream of the 5' side of the protospacer adjacent motif (PAM) sequence (5'-NGG-3'; N is any base) in the cDNA sequence of each target gene were used as the guide sequence. Furthermore, a sequence containing a restriction enzyme site at the Cas9 cleavage site was selected as the guide sequence. The guide sequences set for each gene, their positions on the cDNA, and the restriction enzyme sites at the expected Cas9 cleavage site are summarized in Table 1. Note that cDNA is complementary DNA obtained by reverse transcribing mRNA, and the cDNA sequence corresponds to the mRNA sequence.

[0076]

[0077] Structure of the genome editing vector pGECj (Figure 1) was used as the genome editing vector. pGECj contains a CRISPR / Cas9 unit expressed in cedar, a plant kanamycin resistance marker, an E. coli replication origin, an Agrobacterium replication origin, a spectinomycin resistance marker for E. coli and Agrobacterium, and T-DNA border sequences (LB, RB) for integrating the cassette into plant chromosomes. The guide sequences for each target gene listed in Table 2 were inserted into the "Guide sequence" section shown in Figure 1.

[0078] The genome editing vector into which the guide sequence for the SR10 gene (SEQ ID NO: 25) was inserted was designated pGECj-SR10, the genome editing vector into which the guide sequence for the SR11 gene (SEQ ID NO: 26) was inserted was designated pGECj-SR11, the genome editing vector into which the guide sequence for the SR13 gene (SEQ ID NO: 27) was inserted was designated pGECj-SR13, the genome editing vector into which the guide sequence for the SR14 gene (SEQ ID NO: 28) was inserted was designated pGECj-SR14, the genome editing vector into which the guide sequence for the SR15 gene (SEQ ID NO: 29) was inserted was designated pGECj-SR15, the genome editing vector into which the guide sequence for the SR17 gene (SEQ ID NO: 30) was inserted was designated pGECj-SR17, the genome editing vector into which the guide sequence for the SR24 gene (SEQ ID NO: 31) was inserted was designated pGECj-SR24, and the genome editing vector into which the guide sequence for the SR25 gene (SEQ ID NO: 32) was inserted was designated pGECj-SR25.

[0079] Introduction of genome editing vectors into Agrobacterium. Rhizobium radiobacter GV3101 was used as the Agrobacterium for transformation. pGECj (-SR10, -SR11, -SR13, -SR14, -SR15, -SR17, -SR24, and -SR25) was introduced into GV3101 electro-competent cells (Lifeasible) by electroporation. GV3101 was cultured in YEB medium (5 g / L peptone, 1 g / L yeast extract, 5 g / L beef extract, 5 g / L sucrose, and 0.5 g / L magnesium sulfate heptahydrate) supplemented with 25 μg / mL rifampicin and 25 μg / mL gentamicin. 100 μg / mL spectinomycin was also added to cultures of pGECj-transformed strains. Both the GV3101 strain and its transformants were cultured at 28°C in the dark.

[0080] - Introduction of genome editing cassette into Sugi callus using the Agrobacterium method The procedure for transforming Sugi callus using Agrobacterium was in accordance with "Transformation Protocol [Plant Edition] (Kagaku Dojin) pp. 291-292."

[0081] First, the GV3101 strain carrying the pGECj vector was cultured at OD in a 1 / 2 MD medium supplemented with 50 μM acetosyringone (hereinafter referred to as co-culture medium). 600 The cells were suspended so that the pH was 0.15. 1 g of Japanese cedar callus was added to 20 mL of the above suspension and suspended. This suspension was shaken at 25°C and 120 rpm for 20 minutes. After shaking, 10 mL of the suspension was collected (using a vacuum pump) on sterile filter paper placed in a Buchner funnel. The above filter paper containing the collected cells was placed on top of sterilized filter paper (3 layers) containing 5.5 mL of co-culture medium and cultured (co-culture) in the dark at 28°C.

[0082] After co-cultivation, the callus was suspended in 40 mL of 1 / 2 MD liquid medium, and 1 g of callus was collected as a suspension. The collected callus was washed three times with 40 mL of 1 / 2 MD liquid medium using a centrifuge (150 × g, 1 minute, 25°C).

[0083] The washed callus was suspended in 40 mL of 1 / 2 MD medium containing 10 mg / L meropenem (hereafter referred to as sterilization medium), and 10 mL of the suspension was collected (using a vacuum pump) onto a sterile filter paper placed in a Buchner funnel. The filter paper from which the callus was collected was placed on solid sterilization medium and cultured at 25°C for 3 days, after which the filter paper was transferred to fresh solid sterilization medium and cultured again at 25°C for 2 weeks.

[0084] The callus was then transferred together with the filter paper to 1 / 2MD medium containing 50 mg / L kanamycin and 10 mg / L meropenem (hereafter referred to as selection medium). After transfer, the transformants were cultured on the selection medium until they formed colonies, during which time they were subcultured on fresh selection medium every two weeks. Transformants that grew on the selection medium were picked and used as transformed callus.

[0085] 3. Purification and screening of transformed callus. Transformed callus was suspended in 1 / 2 MD liquid medium to separate individual cells as much as possible, and then adjusted to 50-100 mg cells / 10 mL. 10 mL of the suspension was collected (using a vacuum pump) onto sterile filter paper placed in a Buchner funnel. The filter paper containing the transformed callus was then placed on 1 / 2 MD solid medium supplemented with 2 mM hydrogen peroxide, 30 μM menadione, or 1 μM paraquat and cultured at 25°C. Clones that grew during culture were selected and used as genome-editing candidate strains.

[0086] 4. Confirmation of target gene sequence mutations in genome-edited candidate strains - Extraction of genomic DNA from callus Cell disruption and genomic DNA extraction from genome-edited candidate and parent callus strains were performed using a BioMasher II (Nippon Biomedical Engineering) and the Kaneka Easy DNA Extraction Kit version 2 (Kaneka Biomedical Engineering). After suspending 100 μL of Solution A (included in the Kaneka Easy DNA Extraction Kit version 2) and an equivalent volume of callus to Solution A, the callus was disrupted for approximately 10–20 seconds using the BioMasher. The subsequent DNA extraction steps followed the Kaneka Easy DNA Extraction Kit version 2 procedure.

[0087] PCR amplification of target regions: Using the extracted genomic DNA as a template, PCR was performed using a primer set designed to sandwich the guide sequence region of each target gene (Table 3). MightyAmp DNA Polymerase Ver. 3 (Takara Bio Inc.) was used as the DNA polymerase for PCR, and the denaturation and extension temperatures were set according to the kit's specifications, while the annealing temperature was set according to the Tm value of each primer.

[0088]

[0089] Determining the Presence of Sequence Mutations in the Target Region Using Restriction Enzymes: DNA fragments amplified by PCR using the above primer set were subjected to restriction enzyme digestion to detect the presence of sequence mutations in the target gene due to genome editing. DNA fragments derived from the parent strain are digested by restriction enzymes at the restriction enzyme cleavage sites designed into the guide sequence portion of each gene, but clones that have undergone genome editing will have sequence mutations at the restriction enzyme cleavage sites in the guide sequence, which should prevent the relevant sites from being digested by restriction enzymes. Therefore, clones whose restriction enzyme cleavage patterns in the target region DNA amplified by PCR differ from those of the parent strain can be determined to be clones in which sequence mutations in the target region due to genome editing have occurred.

[0090] To determine the presence or absence of sequence mutations in the target regions of the SR10 and SR25 genes, restriction enzyme TaqI (Takara Bio) was used and digested at 65°C for 3 hours. To determine the presence or absence of sequence mutations in the target regions of the SR13 and SR14 genes, restriction enzyme Hpy188I (New England Biolabs) was used and digested at 37°C for 3 hours. To determine the presence or absence of sequence mutations in the target regions of the SR11 and SR17 genes, restriction enzyme HaeIII (New England Biolabs) was used and digested at 37°C for 3 hours. To determine the presence or absence of sequence mutations in the target region of the SR15 gene, restriction enzyme NlaIII (New England Biolabs) was used and digested at 37°C for 3 hours. To determine the presence or absence of sequence mutations in the target region of the SR24 gene, restriction enzyme AluI (Takara Bio) was used and digested at 37°C for 3 hours. The PCR products of the target region and the PCR products after restriction enzyme treatment were confirmed by agarose gel electrophoresis using TAE buffer (40 mM Tris-acetate, 1 mM EDTA).

[0091] Clones in which sequence mutations were detected in the guide sequence of the target gene were designated as genome-edited strains, with the genome-edited strain in which sequence mutations were detected in the SR10 gene designated #SR10, the genome-edited strain in which sequence mutations were detected in the SR11 gene designated #SR11, the genome-edited strain in which sequence mutations were detected in the SR13 gene designated #SR13, the genome-edited strain in which sequence mutations were detected in the SR14 gene designated #SR14, the genome-edited strain in which sequence mutations were detected in the SR15 gene designated #SR15, the genome-edited strain in which sequence mutations were detected in the SR17 gene designated #SR17, the genome-edited strain in which sequence mutations were detected in the SR24 gene designated #SR24, and the genome-edited strain in which sequence mutations were detected in the SR25 gene designated #SR25.

[0092] The results of confirming sequence mutations in each genome-edited strain are shown in Figure 2 (#SR10), Figure 3 (#SR11), Figure 4 (#SR13), Figure 5 (#SR14), Figure 6 (#SR15), Figure 7 (#SR17), Figure 8 (#SR24), and Figure 9 (#SR25). The electrophoresis patterns of the PCR products after restriction enzyme treatment for each genome-edited strain were different from those of the parent strain, confirming that sequence mutations had occurred in the target region of each gene.

[0093] 5. Evaluation of ROS stress tolerance of genome-edited strains The ROS stress tolerance of genome-edited strains was evaluated by comparing their growth rate when exposed to ROS stress with that of the parent strain. To assess stress tolerance, hydrogen peroxide was used as a direct ROS source, and paraquat and menadione were used as ROS generators.

[0094] As a specific evaluation method, the genome-edited strain and the parent strain were placed on 1 / 2MD solid medium supplemented with hydrogen peroxide (2 mM), menadione (30 μM), or paraquat (1 μM) at concentrations that completely inhibited the growth of the parent strain, and on regular 1 / 2MD medium without an ROS source (hereinafter referred to as the control group), and the growth rates were compared after culturing at 25°C for two weeks.

[0095] The results of growth responses in various media are shown in Figure 10. The parent strain grew normally in the control group (Figure 10-A), whereas growth was completely inhibited in the ROS stress-applied test groups (Figure 10-B, C, D). However, unlike the parent strain, the genome-edited strains also grew in the ROS stress-applied test groups (Figure 10-B, C, D). Therefore, it can be concluded that the #SR10, #SR11, #SR13, #SR14, #SR15, #SR17, #SR24, and #SR25 strains all have improved ROS stress resistance.

[0096] 6. Method for redifferentiating Sugi callus - Induction of somatic embryos from Sugi callus Sugi callus masses were separated into pieces with a diameter of approximately 5 mm and placed on COM medium and cultured at 25°C in the dark for approximately 6 to 8 weeks. The composition of COM medium is shown in Table 4.

[0097]

[0098] Plant regeneration from somatic embryos: Somatic embryos were picked with tweezers and placed on GD medium (20 mm thick Petri dish). After placement on GD medium, they were cultured at 25°C, 16-hour photoperiod, and approximately 2,000 lux of light. After approximately 6-8 weeks of culture, the germinated individuals obtained were transplanted into culture bottles containing GR medium and cultured at 25°C, 16-hour photoperiod, and approximately 2,000 lux of light until they reached a plant size of 2-3 cm or larger. The composition of GD medium is shown in Table 5, and the composition of GR medium is shown in Table 6.

[0099]

[0100]

[0101] The appearance of regenerated individuals obtained by redifferentiation treatment of genome-edited callus and parent callus is shown in Figure 11. The generation of regenerated individuals was confirmed in #SR10, #SR11, #SR14, #SR15, #SR17, #SR24, and #SR25.

[0102] Acclimation to pot culture: After removing plants grown in culture bottles (GR medium), the medium on the plants was washed off and old roots were removed with sterilized scissors. To promote rooting after transplanting, the plants were treated with undiluted Oxyberon (Bayer CropScience) for 10 seconds, ensuring that the area near the root-stem boundary was submerged. After treatment with Oxyberon, the plants were transplanted into pots containing soil containing a mixture of coco peat, rice husks, and charcoalized rice husks in an 80:20:3 ratio. After thoroughly moistening the soil, the pots were placed in resealable bags to prevent drying. The transplanted seedlings were then grown in the resealable bags at 25°C, with a 16-hour photoperiod and approximately 2,000-5,000 lux of light for approximately three weeks to acclimate them to pot culture.

[0103] According to the present invention, the ROS tolerance of plants can be improved and the CO ２ The amount of immobilization can be increased. Therefore, the present invention is useful for solving global environmental and energy problems.

[0104] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

[0105] SEQ ID NO:25: Guide sequence (SR10) SEQ ID NO:26: Guide sequence (SR11) SEQ ID NO:27: Guide sequence (SR13) SEQ ID NO:28: Guide sequence (SR14) SEQ ID NO:29: Guide sequence (SR15) SEQ ID NO:30: Guide sequence (SR17) SEQ ID NO:31: Guide sequence (SR24) SEQ ID NO:32: Guide sequence (SR25) SEQ ID NO:33: Forward primer (SR10) SEQ ID NO:34: Reverse primer (SR10) SEQ ID NO:35: Forward primer (SR11) SEQ ID NO:36: Reverse primer (SR11) SEQ ID NO:37: Forward primer (SR13) SEQ ID NO:38: Reverse primer (SR13) SEQ ID NO:39: Forward primer (SR14) SEQ ID NO:40: Reverse primer (SR14) SEQ ID NO:41: Forward primer (SR15) SEQ ID NO:42: Reverse primer (SR15) SEQ ID NO:43: Forward primer (SR17) SEQ ID NO:44: Reverse primer (SR17) SEQ ID NO: 45: Forward primer (SR24) SEQ ID NO: 46: Reverse primer (SR24) SEQ ID NO: 47: Forward primer (SR25) SEQ ID NO: 48: Reverse primer (SR25)

Claims

1. A plant cell having resistance to reactive oxygen species (ROS), the plant cell comprising a modification that reduces the function of at least one of the following endogenous genes (a) to (c): (a) a gene comprising a nucleotide sequence shown in any one of SEQ ID NOs: 1 to 8; (b) a gene comprising a base sequence having 80% or more sequence identity with any of the base sequences shown in SEQ ID NOs: 1 to 8, the reduction in function of which confers ROS resistance in a plant; (c) a gene that hybridizes under stringent conditions with a gene comprising a nucleotide sequence shown in any one of SEQ ID NOs: 1 to 8, and whose reduced function confers ROS resistance in a plant.

2. The plant cell according to claim 1 , wherein the modification resulting in reduced function of the endogenous gene is a mutation in a genomic sequence resulting in loss of function of the endogenous gene.

3. The plant cell according to claim 1 , wherein the modification resulting in reduced function of the endogenous gene is a mutation that reduces or destroys expression of the endogenous gene.

4. The plant cell of claim 1 , wherein the modification resulting in reduced function of the endogenous gene is one or more indels in the ORF region of the endogenous gene.

5. A plant or a part thereof, which is constituted by the plant cell according to any one of claims 1 to 4.

6. 6. The plant body or a part thereof according to claim 5, wherein the part of the plant body is any one selected from the group consisting of callus, plant tissue, plant tissue culture, seeds, fruits, leaves, stems, trunks, roots, and flowers.

7. 7. A plant or a part thereof according to claim 5 or 6, in which the amount of CO2 fixed by the plant is increased.

8. The plant cell according to any one of claims 1 to 4, or the plant or part thereof according to any one of claims 5 to 7, wherein the plant is any one plant selected from the Cupressaceae family, Pinaceae family, Myrtaceae family, and Betulaceae family.

9. A method for producing a plant with an increased amount of CO2 fixation, comprising obtaining a plant cell containing a modification that reduces the function of at least one reactive oxygen species (ROS) resistance-related gene, and generating a plant body from the plant cell; The method, wherein the ROS resistance-related gene is any one of the following (a) to (c): (a) a gene comprising a nucleotide sequence shown in any one of SEQ ID NOs: 1 to 8; (b) a gene comprising a base sequence having 80% or more sequence identity with any of the base sequences shown in SEQ ID NOs: 1 to 8, the reduction in function of which confers ROS resistance in a plant; (c) a gene that hybridizes under stringent conditions with a gene comprising a nucleotide sequence shown in any one of SEQ ID NOs: 1 to 8, and whose reduced function confers ROS resistance in a plant.

10. A method for increasing the amount of CO2 fixed in a plant body, comprising modifying at least one reactive oxygen species (ROS) resistance-related gene to reduce its function, and generating a plant body from the plant cell; The method, wherein the ROS resistance-related gene is any one of the following (a) to (c): (a) a gene comprising a nucleotide sequence shown in any one of SEQ ID NOs: 1 to 8; (b) a gene comprising a base sequence having 80% or more sequence identity with any of the base sequences shown in SEQ ID NOs: 1 to 8, the reduction in function of which confers ROS resistance in a plant; (c) a gene that hybridizes under stringent conditions with a gene comprising a nucleotide sequence shown in any one of SEQ ID NOs: 1 to 8, and whose reduced function confers ROS resistance in a plant.

11. The method according to claim 9 or 10, wherein the plant is any plant selected from the Cupressaceae family, Pinaceae family, Myrtaceae family, and Betulaceae family.