Plant with high oleic acid containing seed and production method thereof

Abstract

The present invention aims to provide a plant body that produces high-oleic seeds while mitigating the side effects of growth defects, and a method for producing the same.

Description

Technology Field

[0001] The present invention relates to a plant body that produces high-oleic acid containing seeds and a method for producing the same. Background Technology

[0002] Vegetable oil is oil extracted from plant seeds or fruits, and it is widely used for edible purposes, with demand steadily increasing. In order to improve storage stability and prevent rancidity, it is preferred to lower the degree of unsaturation of the fatty acids contained in vegetable oil.

[0003] In this regard, since the FAD2 (Fatty Acid Desaturase 2) gene in plants plays a role in converting oleic acid, a monounsaturated fatty acid, into linoleic acid, a polyunsaturated fatty acid, there have been attempts to obtain seeds with increased oleic acid content by inhibiting the function of the FAD2 gene.

[0004] For example, there have been cases where the FAD2 gene was knocked out using activator-like effector nucleases (TALENs) in peanuts and soybeans, or where the gene was knocked out using CRISPR / Cas9 in peanuts, soybeans, and camellias; however, FAD2 gene mutations obtained from conventional technology exhibit growth defects in plants, making it difficult to utilize them for agronomic purposes.

[0005] In this regard, Korean Patent No. 2117110 also describes a mutation in the FAD2 gene that increases the oleic acid content of soybeans (Glycine max L.) using CRISPR-Cas9, but does not describe the effect of improving growth defect side effects.

[0006] Therefore, there is an urgent need to develop new plant bodies that produce seeds with increased oleic acid content without these side effects. Prior art literature

[0008] Korean Patent No. 2117110 The problem to be solved

[0009] The present invention aims to provide a new transgenic plant containing high oleic acid seeds and a method for producing the same. means of solving the problem

[0011] 1. A plant expressing a FAD2 protein, wherein the protein is composed of a sequence corresponding to the sequence of SEQ ID NO. 1, and the amino acid at the position corresponding to the 295th position in SEQ ID NO. 1 is Gly or Val.

[0012] 2. In the above 1, the protein is a plant in which the amino acid at the position corresponding to the 295th position in SEQ ID NO. 1 is Gly and the amino acid at the position corresponding to the 298th position is Glu.

[0013] 3. In the above 1, the protein is a plant in which the amino acid at the position corresponding to the 295th position in SEQ ID NO. 1 is Val, and the amino acid at the position corresponding to the 296th position is Met.

[0014] 4. In the above 1, the plant body is a plant body that is sesame, sesame, perilla, peanut, rice bran, soybean, rapeseed, flax, castor oil plant, palm tree, olive, camellia, safflower, hemp, flax, paulownia, sunflower, olive, peanut, camellia, castor oil plant, rapeseed, Arabidopsis thaliana, hazelnut tree, red eucalyptus, rice, resquerella, castor oil plant, or corn.

[0015] 5. Seeds produced by the plant body of 1 above.

[0016] 6. A method for producing a plant, comprising the step of modifying a gene encoding a FAD2 protein, which is composed of a sequence corresponding to the sequence of SEQ ID NO. 1, so that the amino acid at the position corresponding to the 295th position in SEQ ID NO. 1 is substituted with Gly or Val.

[0017] 7. A method for producing a plant according to 6 above, further comprising the step of selecting a plant that expresses a protein in which the amino acid at the position corresponding to the 295th position in SEQ ID NO. 1 is Gly and the amino acid at the position corresponding to the 298th position is Glu.

[0018] 8. A method for producing a plant, wherein, in the above 6, among the mutated plant bodies, the amino acid at the position corresponding to the 295th position in SEQ ID NO. 1 is Val, and the amino acid at the position corresponding to the 296th position is Met.

[0019] 9. A method for producing a plant in which the above substitution is performed using gRNA of SEQ ID NO. 2, in accordance with 6 above. Effects of the invention

[0021] The plant of the present invention can be used in the plant maintenance business field because it produces high-oleic acid containing seeds while mitigating growth defects compared to cases where the FAD2 gene is knocked out.

[0022] By using the method of the present invention, it is possible to produce high-oleic acid containing seeds while producing plants with reduced growth defects compared to cases where the FAD2 gene is knocked out. Brief explanation of the drawing

[0024] Figure 1 compares the FAD2 amino acid sequences of Arabidopsis thaliana and other oil crops, and protein alignment was performed using ClustalW of the MEGA7 program centered on the g5 region. The red box indicates the base editing window in this invention. At is Arabidopsis thaliana , Ca is Corylus avellana , Ec is Eucalyptus camaldulensis , Gm is Glycine max , Os is Oryza sativa , Pf is Physaria fendleri , Rc is Ricinus communis , Zm is Zea mays It means. Figure 2 compares the nucleotide sequences of the FAD2 gene of oil crops other than Arabidopsis thaliana, showing that the 2nd cytosine and 12th cytosine regions of Arabidopsis thaliana, which were base-edited in the present invention, are preserved in other crops as well. In FIG. 3, a is a schematic representation of the function of FAD2, the base-editing target of the present invention, in fatty acid unsaturation. FAD2 is located in the endoplasmic reticulum (ER) and contains six transmembrane domains and three histidine box motifs (His). b shows the core structure of the CRISPR portion in the T-DNA of the binary vector containing CBE and ABE in the present invention. c is a schematic diagram of the FAD2 protein structure. Red dots indicate the base-editing target regions. d shows the amino acid sequence of the Arabidopsis FAD2 protein. The green sequences represent the transmembrane regions, the red sequences represent the His motifs, and the red lines represent potential amino acids affected by base-editing targeting via the corresponding gRNAs. e shows a list of selected gRNAs used in CBE and / or ABE containing protospacer sequences for FAD2 editing in the present invention. Figure 4 illustrates the pJY-RpAID binary vector. Figure 5a shows the Sanger sequencing results for the FAD2 genotype within the editing window of the T2 plant. The editing window is shown in the upper Col-0 panel, and red characters indicate modified DNA and amino acid sequences. Figure 5b is a table analyzing the fatty acid composition of the transformed lines. Red numbers indicate the cases where the highest oleic acid content was found among the analyzed T2 plants. * indicates a representative T2 transformed line that has progressed to the T3 generation. Figure 6 shows the sequencing results of T1 plants g5CBE11 to 17 and g5CBE31 to 35. The red arrow indicates the changed sequence in the FAD2 gene. Figure 7a summarizes the T3 isolated patterns and presents the analysis of oleic acid content according to each genotype. Characters in green and gray shading represent the protospacer sequence and PAM sequence, respectively. Red characters represent the altered DNA sequence. Figure 7b is a chromatogram showing the Sanger sequencing results for FAD2 genotype analysis within the FAD2 allele editing window. Red characters represent the altered DNA and amino acid sequences. Figure 7c compares the fatty acid composition among T4 seeds of the new FAD2 allele, FAD2-1, and Col-0. Differences between Col-0 and FAD2 alleles were identified using two-way ANOVA statistical analysis (*** p < 0.001), the value represents the mean ± sd. Figure 8 shows the Sanger sequencing results of the FAD2 gene in T3 plants. * indicates a new representative allele isolated in the present invention. Figures 9 and 10 show the results of the physiological response analysis of the weakened FAD2 allele. Figure a shows the results of germination tests for the FAD2 allele performed under ½ MS, b under ½ MS; 150 mM NaCl, and c under MS; 300 mM mannitol. Figure ac is a photograph taken 96 hours after sowing, conducted to test whether an increase in oleic acid content affects germination under both normal and stress conditions. Germination rates were measured every 12 hours and performed in triplicate. Figures d through f show the results of root growth rate analysis of the FAD2 allele under normal and stress conditions, in which seedlings were grown at ½ MS for 4 days and root development occurred over 5 days. d is a photograph taken to identify the root length of seedlings grown for 5 days under experimental conditions, e is a graph showing root length under various conditions, and f is a graph showing relative root length under 75 mM NaCl and 200 mM mannitol conditions. Root length was set to 100% at ½ ms, and all values ​​were expressed as mean ± sd. Specific details for implementing the invention

[0025] In this invention, various genetic modification sites were selected based on the overall structure of the FAD2 protein, and various genetic modifications were attempted at each site to obtain a plant with "weakened" function of the FAD2 protein.

[0026] In the present invention, a plant with weakened function of the FAD2 protein refers to a plant in which the function of the FAD2 protein has not been completely lost; for example, it refers to a plant in which some functions of the FAD2 protein are weakened, while other functions are weakened or maintained.

[0027] In other words, in order to obtain a plant in which the gene encoding the FAD2 protein is not simply knocked out, sophisticated amino acid modifications were performed targeting various locations of the FAD2 protein. As a result, the oleic acid conversion function of the FAD2 protein was weakened to produce seeds containing high oleic acid, while the resistance function of the FAD2 protein to salt stress was not completely lost, thereby obtaining a plant with improved side effects of growth defects and completing the invention.

[0029] The present invention will be described in detail below.

[0031] The present invention relates to a plant that expresses a FAD2 protein, wherein the protein is composed of a sequence corresponding to the sequence of SEQ ID NO. 1, and the amino acid at the position corresponding to the 295th position in SEQ ID NO. 1 is Gly or Val.

[0032] The above FAD2 (Fatty Acid Desaturase 2) protein is a type of fatty acid desaturase enzyme, and in plants, the unsaturated fatty acid oleic acid (18:1 Δ9 ) linoleic acid (18:2 Δ9,12 It is involved in synthesizing into ).

[0033] The above "fatty acid" refers to a hydrocarbon chain having one carboxyl group (-COOH). Most fatty acids in their natural state have an even number of carbons, ranging from about 4 to 36, and fatty acids can be classified into saturated or unsaturated fatty acids depending on whether the chain is saturated (or has carbon-carbon double bonds).

[0034] The above "saturated fatty acid" refers to a fatty acid composed of single bonds, and "unsaturated fatty acid" refers to a fatty acid containing one or more carbon-carbon double bonds. Unsaturated fatty acids can be classified into omega-3 fatty acids, omega-6 fatty acids, omega-7 fatty acids, and omega-9 fatty acids depending on the position where the carbon-carbon double bond begins; for example, omega-9 fatty acids refer to unsaturated fatty acids in which the double bond begins at the ninth carbon from the end of the carbon chain.

[0035] Vegetable oil contains two saturated fatty acids, palmitic acid (16:0) and stearic acid (18:0), and three types of unsaturated fatty acids, oleic acid (18:1 Δ9 ), linoleic acid (18:2 Δ9,12 ) and linolenic acid (18:3 Δ9,12,15 ) is included.

[0036] The aforementioned "oleic acid" is an omega-9 unsaturated fatty acid, mostly found in animal and vegetable oils, and is the main component of oils such as camellia oil and olive oil.

[0037] In the present invention, FAD2 is involved in the synthesis of linoleic acid (18:2 Δ9,12 ) is an omega-6 fatty acid and is one of the essential fatty acids that humans must obtain through food.

[0038] By inhibiting the function of the above-mentioned FAD2 in the plant body, the oleic acid content of the produced seeds can be increased, and since the above-mentioned linoleic acid has the property of being easily oxidized compared to oleic acid, vegetable oil produced from seeds containing high oleic acid has high storage stability.

[0039] Meanwhile, in plants, the activity of the FAD2 protein is associated with an increase in the content of dienic acid, which increases resistance to cold and salt stress. Since it is involved in the salicylic acid (SA), oxidase, abscisic acid, and jasmonic acid (JA) pathways, simply knocking out the FAD2 protein results in growth defects.

[0040] Sequence No. 1 represents the full-length FAD2 protein sequence of Arabidopsis, a model plant used in the embodiment of the present invention.

[0041] The "corresponding sequence" above refers to a sequence corresponding to another sequence when the two sequences are aligned, and the "corresponding position" above refers to a position corresponding when the sequence is aligned with the corresponding sequence. For example, as shown in FIGS. 1 and 2 of the present invention, the FAD2 gene or protein sequence of Arabidopsis thaliana can be aligned with the nucleotide sequence or protein sequence of the FAD2 gene of various plant species to identify the corresponding position. The FAD2 protein sequence is a sequence with high conservation among plant species, and specifically, FIGS. 1 and 2 can be confirmed that the 295th, 296th, and 298th amino acids A, T, and D of Sequence No. 1, which is the subject of base editing in the present invention, are conserved identically in various plants.

[0042] The above plant body may be sesame, sesame, perilla, peanut, rice bran, soybean, rapeseed, flax, castor bean, palm tree, olive, camellia, safflower, hemp, flax, paulownia, sunflower, olive, peanut, camellia, castor bean, rapeseed, Arabidopsis thaliana, hazelnut tree, red eucalyptus, rice, resquerella, castor bean, or corn, but is not limited thereto.

[0043] The present invention comprises a plant in which the amino acid at the position corresponding to the 295th position in SEQ ID NO. 1 is Gly, and the amino acid at the position corresponding to the 298th position is Glu.

[0044] The present invention comprises a plant in which the amino acid at the position corresponding to the 295th position in SEQ ID NO. 1 is Val, and the amino acid at the position corresponding to the 296th position is Met.

[0045] The present invention includes seeds produced by the above-mentioned plant body.

[0046] The above plant is a plant that expresses FAD2 protein, and means a plant in which the amino acid at the position corresponding to the 295th position in the FAD2 protein sequence is Gly or Val, a plant in which the amino acid at the position corresponding to the 298th position in SEQ ID NO. 1 is Glu when the amino acid at the position corresponding to the 295th position in SEQ ID NO. 1 is Gly, or a plant in which the amino acid at the position corresponding to the 296th position in SEQ ID NO. 1 is Met when the amino acid at the position corresponding to the 295th position in SEQ ID NO. 1 is Val.

[0047] The above seeds have a high proportion of oleic acid. For example, wild-type seeds generally have an oleic acid content of 20 wt.% or less of the total fatty acid weight of the seeds, but seeds produced from the plant body of the present invention had a significantly higher oleic acid content of 30 to 65 wt.%.

[0048] In addition, the present invention includes a method for producing a plant, comprising the step of modifying a gene encoding a FAD2 protein having a sequence corresponding to the sequence of SEQ ID NO. 1 in a plant so that the amino acid at the position corresponding to the 295th position in SEQ ID NO. 1 is substituted with Gly or Val.

[0049] The above plant is not particularly limited to any plant that expresses the FAD2 protein, and may be, for example, sesame, sesame, perilla, peanut, rice bran, soybean, rapeseed, flax, castor bean, palm tree, olive, camellia, safflower, hemp, flax, paulownia, sunflower, olive, peanut, camellia, castor bean, rapeseed, Arabidopsis thaliana, hazelnut, red eucalyptus, rice, resquerella, castor bean, or corn.

[0050] The above gene mutation may, for example, modify the base sequence of DNA or RNA encoding the FAD2 protein, and the method of modification is not particularly limited as long as it is a method that can be appropriately selected by a person skilled in the art.

[0051] The above “base sequence” refers to a sequence of bases arranged in order, which are one of the constituent components of nucleotides, the basic units of DNA or RNA. When the bases are arranged in a line in groups of three, they form a triplet code that specifies one amino acid. Since multiple triplet codes together ultimately specify one protein, when bases are arranged in groups of three, they form a triplet code and are converted into a protein sequence. Therefore, for example, the 295th amino acid Ala of Sequence No. 1 can be substituted with Gly by modifying the DNA base sequence “GCT” coding for it to “GGT”, or substituted with Val by modifying it to “GTT”. Additionally, the 296th amino acid Thr of Sequence No. 1 can be substituted with Met by modifying the DNA base sequence “ACC” coding for it to “ATG”, and the 298th amino acid Asp of Sequence No. 1 can be substituted with Glu by modifying the DNA base sequence “GAC” coding for it to “GAG” or “GAA”.

[0052] The modification of the above base sequence may be due to a "transformation" method. Transformation refers to the process in which a vector, such as a DNA fragment or plasmid containing a type of gene different from that of the original cell, infiltrates the cell and binds to the DNA present in the original cell, thereby altering the genetic traits.

[0053] Plant transformation refers to any method of transferring DNA into a plant. Any transformation method can be used to introduce recombinant DNA according to the present invention into a suitable progenitor cell. For example, the calcium / polyethylene glycol method on protoplasts (Krenset et al., 1982, Nature 296: 72-74; Negrutiu et al., 1987, Plant Mol. Biol. 8: 363-373), electroporation of protoplasts (Shillito et al., 1985, Bio / Technol. 3: 1099-1102), microinjection into plant elements (Crossway et al., 1986, Mol. Gen. Genet. 202: 179-185), particle impaction of various plant elements (DNA or RNA-coated) (Klein et al., 1987, Nature 327: 70), viral infection (EP 0) in Agrobacterium tumopaciens-mediated gene transfer (incompleteness) via plant infiltration or transformation of mature pollen or microspores It can be appropriately selected from No. 301316, etc.

[0054] The “plant cells” used for the transformation of the above-mentioned plant are not particularly limited, and the plant cells may be cultured cells, cultured tissues, cultured organs, or cells of the whole plant, and the tissues include differentiated or undifferentiated plant tissues, e.g., roots, stems, leaves, pollen, seeds, female tissues, and various forms of cells used for culture, namely single cells, protoplasts, buds, and callus tissues. The plant tissues may be in planta or in organ culture, tissue culture, or cell culture.

[0055] The above transformation may include the step of transforming plant cells using a recombinant vector and redifferentiating transformed plant cells from the transformed plant cells. Any method known in the art may be used for redifferentiating transformed plant cells from transformed plant cells (Perez-Garcia, P., Moreno-Risueno, MA (2018) Stem cells and plant regeneration. Developmental Biology, 442: 3-12, Ikeuchi, M., Ogawa, Y., Iwase, A. et al. (2016) Plant regeneration: cellular origins and molecular mechanisms. Development (Cambridge, England), 143: 1442-51).

[0056] The above "recombinant vector" refers to a vector manufactured to express a target protein in a suitable host cell, and is a gene construct containing essential regulatory elements operably linked to express a gene insert. In addition, in the present invention, a binary vector is a vector designed to function across two vector systems. Since the Ti-plasmid of Agrobacterium used for plant transformation is difficult to manipulate due to its size, it is divided into two replicons; one contains vir genes and the remaining genes are located in the other replicon to facilitate manipulation.

[0057] Vectors that can be used in the present invention include plasmid vectors, cosmid vectors, bacteriophage vectors, and viral vectors such as adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors. Suitable expression vectors may include signal sequences or leader sequences for membrane targeting or secretion in addition to expression regulatory elements such as promoters, start codons, stop codons, polyadenylation signals, and enhancers, and may be prepared in various ways depending on the purpose.

[0058] In the present invention, the recombinant vector may be a gene editing technology, such as BEs (Base Editors), zinc finger nucleases (ZFNs), TALENs, CRISPR / Cas9, etc., but is not limited thereto.

[0059] Preferably, the aforementioned base substitution can be performed using Base Editors (BEs), a technology developed from CRISPR / Cas9. Since the BEs can induce C-to-T (substitution of cytosine with thymine) or A-to-G (substitution of adenine with guanine) base editing at the target site without two-strand breaks (DSBs), they are widely used as a method for inducing precise mutations at the target site.

[0060] BEs are technologies developed by fusing Nickase Cas9 (nCas9) with cytosine deaminase or adenine deaminase, which are called cytosine base editors (CBE; conversion C to T) or adenine base editors (ABE; conversion A to G), respectively. BEs can cause changes in amino acids or modifications in splicing, and for the two CBE systems, base editor 3 (BE3) and activation-induced cytidine deaminase (AID), rat rAPOBEC1 and sea lamprey PmCDA1 were applied as cytidine deaminase domains.

[0061] The target site for the aforementioned gene editing can be specified by "gRNA (guide RNA)." gRNA is an RNA that acts as a guide to determine which part of DNA to modify; since it is capable of complementary binding to the target sequence, CRISPR can bind to the target sequence by utilizing the characteristics of this RNA.

[0062] For the above CRISPR gene scissors to function, a PAM (Protospacer-Adjacent Motif) sequence may be required adjacent to the target sequence, and the PAM sequence may be in the form of NGG (where N represents any base sequence among A, T, G, and C).

[0063] The aforementioned "CRISPR / Cas9" is a type of third-generation gene editing tool composed of "gRNA" and "Cas9," an enzyme that cuts DNA. CRISPR is a DNA sequence found in the genomes of prokaryotic organisms such as bacteria and archaea. Cas9 is an enzyme that uses the CRISPR sequence as a guide to recognize and cut specific strands of DNA complementary to the CRISPR sequence. In this process, gRNA forms a complex with Cas9, a restriction enzyme that cuts DNA double helix. When this enzyme is inserted into the region where the gene is to be manipulated, it locates the target DNA sequence, and Cas9 cuts the DNA. Cells possess a repair mechanism when DNA is cut. If this process is repeated, "repair errors" occur, resulting in a difference of several bases from the original sequence. When such a difference arises, CRISPR stops functioning, and the sequence altered by the repair error is unable to perform its original function.

[0064] In this way, it becomes possible to accurately locate and knock out the gene to be cut, and this technology can also be used to add genes to desired locations in addition to cutting. When a new DNA sequence to be added is inserted along with CRISPR / Cas9, the cell absorbs the added DNA sequence during the process of repairing the cut area.

[0065] Consequently, since CRISPR / Cas9 is highly likely to result in randomly formed insertions or deletions (indels) at the target site, causing loss-of-function alleles (gene knockout), the embodiments of the present invention performed transformation using the aforementioned BEs.

[0066] In the present invention, the above substitution may be performed using the gRNA of SEQ ID NO. 2. Since the description of the gRNA has been previously provided, it is omitted to avoid duplication. Specifically, the gRNA of SEQ ID NO. 2 can bind complementarily to the gene encoding the 295th to 298th amino acids of the FAD2 protein, and thus can induce a modification of the amino acid position targeted in the present invention.

[0067] The above gRNA can be used in a recombinant vector introduced for gene editing, for example, as a single guide RNA (sgRNA) of the vector shown in FIG. 3b of the present invention. In the above vector, the expression of the gRNA is controlled by the U6 promoter, and the AIDv2-dependent CBE and ABE7.10 are controlled by the RPS5A promoter.

[0068] In addition, the present invention may include the step of selecting a plant among the mutated plants in which the amino acid at the position corresponding to the 295th position in SEQ ID NO. 1 is Gly and the amino acid at the position corresponding to the 298th position is Glu expresses a protein.

[0069] In addition, the present invention may include the step of selecting a plant that expresses a protein in which the amino acid at the position corresponding to the 295th position in sequence number 1 is Val and the amino acid at the position corresponding to the 296th position is Met among the mutated plant.

[0070] The above screening method is not particularly limited as long as it is a method that can be appropriately selected by a person skilled in the art, for example, it may involve detecting the target plant using a fragment complementary to the gene or protein that has undergone the aforementioned substitution, or screening the gene or protein that has undergone such substitution by a sequence analysis method of the target plant.

[0071] For example, the detection method may utilize Southern blot, Northern blot, Western blot, fluorescence in situ hybridization, etc., and the sequence analysis method may utilize Sanger sequencing, NGS sequencing, Maxam-Gilbert sequencing, etc., but is not limited thereto.

[0072] In one embodiment of the present invention, to confirm the nucleotide sequence of a transformed plant, a Sanger sequencing method was used, which reads the process in which complementary bases paired with the template DNA sequence bind one by one to a new DNA strand during replication based on the DNA polymerase reaction that proceeds during DNA replication.

[0074] The present invention will be explained in more detail below through the following examples. The following examples are merely illustrative of the present invention and do not limit the scope of the present invention.

[0076] Experimental method

[0077] 1. Plant materials and growth conditions

[0078] All experiments were performed using Arabidopsis thaliana Columbia-0 (Col-0) and FAD2-1 as controls. FAD2-1 refers to a control group in which the FAD2 gene induced by EMS (Ethyl Metane Sulphonate) treatment was mutated. Arabidopsis thaliana seeds were disinfected with 70% EtOH and 0.5% NaOCl, and then washed 7–8 times with distilled water before planting. The seeds were stratified at 4°C for 3 days. The seeds were grown in ½ MS media containing 1% sucrose in a tissue culture incubator with a 16 h light / 8 h dark cycle at 23°C.

[0080] 2. Composition of Plasmids

[0081] To construct the underlying CBE binary vector pJY-RpAID, the coding sequence PmCDA1, optimized for the codons of the plant *Arabidopsis thaliana*, was synthesized using Integrated DNA technology (Iowa, USA). First, pKI1.1R (Addgene #85808) was used to create the pJY-RpEmpty vector, in which the original Cas9 sequence was removed and replaced with the positions of two enzymes, XmaJI and XhoI. An in-fusion cloning method (Takara, Japan) was performed on the XmaJI / XhoI-treated pJY-RpEmpty vector, and the Nickase Cas9 (D10A) and the codon-optimized AID were PCR-amplified to create the pJY-RpAID binary vectors. The pJY-RpAID vector contains the following factors from pKI1.1R instead of the original Cas9 expression cassette: RPS5A promoter, SV40 NLS, nCas9 (D10A), SV40 NLS, 67aa linker, 3X FLAG, PmCDA, SV40 NLS, UGI, and heat shock protein 18.2 terminator (Figs. 3b and 4). For the plant transformation vector, Aar1-modified gRNA cloning was performed as previously described. For the FAD2 gene editing of Arabidopsis thaliana, six different 20-mer guide sequences (Fig. 3e) were designed using the CRISPR RGEN Tool (http: / / www.rgenome.net) and cloned into AarI-treated pJY-RpAID or pJY-RpABE vectors.

[0083] 3. Transformation of Arabidopsis and Selection of Transformed Plants

[0084] A. thaliana was transformed by floral dipping using the Agrobacterium strain GV3101. The resulting T1 seeds were germinated in ½ MS media containing 1% sucrose and 50 ng·μL-1 hygromycin. Only plants that survived in the media were selected, transferred to soil, and subjected to subsequent analysis. Genomic DNA was extracted from the leaves for Sanger sequencing, and the occurrence of base editing in the FAD2 gene was analyzed.

[0086] 4. Sanger Sequencing

[0087] Using the extracted genomic DNA as a template, PCR was performed using Ex Taq (Takara) to obtain FAD2 amplicons for subsequent sequencing analysis. Using the forward primer 5'-GCATTGTTTCAAACGCTCAA and the reverse primer 5'-TCATAACTTATTGTTGTACCAGTAC, 30-cycle PCR was performed under the following conditions: pre-denaturation (95°C for 5 min); denaturation (95°C for 30 sec); annealing (52°C for 30 sec); and extension (72°C for 1 min); post-extension (72°C for 10 min). A purification kit (Cosmo Genetech) was used to purify the PCR products. The quality and quantity of the products were verified using Nano drop (Denovix) before being sent to a sequencing company (Bioneer).

[0089] 5. Fatty Acid Analysis

[0090] At least 100 seeds were used for gas chromatography (GC) analysis for each sample. A 5% sulfuric acid solution dissolved in methanol and toluene were prepared. Each sample immersed in the solution was heated in a water bath at 85°C for two hours. Subsequently, 1 mL of 0.9% NaCl and 1 mL of hexane were added to each sample, followed by centrifugation at 330 x g for 2 minutes. The supernatant was transferred to a 6 mL tube and purified using a nitrogen concentrator (Eyela). The extracted fatty acid methyl ester (FAME) was dissolved in 200 µL of hexane and then inserted into a GC vial. A DB-23 column (30 m x 0.25 mm, 0.25 µm film, Agilent) was used, and the extracted FAME was analyzed using a GC-2030 (Shimadzu). The temperature of the GC oven was raised from 190°C to 230°C at 3°C ​​min-1.

[0092] 6. Measurement of germination rate

[0093] Germination tests were performed on seeds stratified at 4°C for 3 days. Germination was recorded when roots began to appear in ½MS media, which consisted of 150 mM NaCl containing 1% sucrose and 300 mM Mannitol containing 1% sucrose. Germination tests were performed every 12 hours for 4 days and repeated 3 times for 40 seeds per series.

[0095] 7. Root growth measurement

[0096] Col-0 and FAD2 alleles containing FAD2-1 were grown under normal and stress conditions, and root development was monitored. Under stress conditions, seedlings 4 days after germination were transferred to ½ MS media containing 75 mM NaCl or 200 mM Mannitol. Root length was measured four times for each of the five individuals. Root length was measured on the fifth day after growing the seedlings perpendicular to the light. Length measurements were performed using the Image J program. Relative root growth (%) was calculated as (average in stress condition MS media) / (average in MS media) x 100.

[0098] 8. Statistical analysis of data

[0099] One-way ANOVA using GraphPad Prism was performed to analyze the differences between the wild-type (Col-0), FAD2-1, and CBE lines. * indicates significant differences compared to wild-type plants (* p < 0.05; ** p < 0.01; *** p < 0.001).

[0101] Experimental results

[0102] 1. Formation of a weakened FAD2 allele through base editing

[0103] The present invention aims to induce a potential "weakened" FAD2 allele by performing CBE or ABE on a plant to induce arbitrary C-to-T and / or A-to-G base substitutions targeting several coding regions of the FAD2 gene, wherein the "weakened" FAD2 allele implies an increase in the content of oleic acid instead of linoleic acid while minimizing side effects such as growth defects. This is based on the principle that when a missense mutation is induced using base editing at a specific location of the FAD2 gene, the structural integrity of the FAD2 gene is maintained, so the function of FAD2 will be weakened rather than completely destroyed (Fig. 3a).

[0104] To this end, the underlying CBE binary vector pJY-RpAID was first designed, and the vector is under the control of the Arabidopsis U6 promoter, the RPS5A promoter, and the gRNA cloning cassette (Figs. 3b and 4).

[0105] In the present invention, six independent gRNAs named "g1 to 6" were designed to perform base editing targeting selected coding regions (Fig. 3e), and the selected coding regions include the N-term transmembrane part, tandem His-motif part, membrane-proximal cytosolic part, cytosolic stretches, and C-term part of the FAD2 protein (Figs. 3c and 3d). As a result of the experiment, five ABEs and three CBEs were introduced into Arabidopsis Col-0 (Fig. 1e). During selective media culture, 26 T1 plants were selected to possess the BE transgene, and they were transferred to soil to perform GC analysis on each of their T2 seeds.

[0106] The genetic source of each T2 seed was obtained from T1 plants, and parallel GC analysis was performed to determine if there were significant changes in lipid composition. The GC analysis results showed that oleic acid levels consistently increased in the T2 seeds of four T1 plants (designated as g5CBE1 to g5CBE4) that underwent CBE using g5 guide RNA. The oleic acid content of g5CBE1, g5CBE2, g5CBE3, and g5CBE4 increased to 36.5%, 36.2%, 48.9%, and 21.6%, respectively, whereas the oleic acid content in the T2 seeds of wild-type Col-0 and other transgenic lines did not exceed 20%. Furthermore, in the case of g5CBE plants, along with the increase in oleic acid, the levels of polyunsaturated fatty acids decreased, and eicocenate (20:1 11 Since it was found that ) increased, this suggests that the incoding enzyme activity of FAD2 protein was mediated in the above transgenic plants (Tables 1 and 2).

[0107] Fatty acid composition (%) of T2 seeds of selected transgenic alleles fatty acid Col-0 Transformation line (n=T1 allele number) g2ABEn g3ABEn g4ABEn g5ABEn g6ABEn 1 2 n=1 1 2 1 2 3 4 1 2 3 1 2 16:0 13.9 13.1 8.7 8.4 7.9 13.0 14.5 13.1 14.1 8.5 9.0 8.8 13.6 9.0 18:0 11.0 9.9 3.6 3.3 3.4 9.1 11.6 9.9 11.6 3.5 3.4 3.7 9.9 3.6 18:1 17.7 18.4 12.1 17.0 19.4 16.1 17.5 18.2 17.9 15.2 13.0 15.1 15.7 13.6 18:2 24.6 26.2 29.3 29.5 26.8 28.1 25.0 25.9 23.6 28.7 29.4 30.2 25.9 29.5 18:3 15.7 16.0 21.6 18.9 18.5 15.8 14.8 15.7 14.8 20.2 21.5 20.0 16.5 20.7 20:0 2.8 1.9 3.2 2.5 2.7 2.0 2.0 1.9 1.8 3.0 2.7 2.9 2.0 3.0 20:1 14.3 14.6 21.5 20.4 21.3 15.9 14.6 15.2 16.2 20.9 20.8 19.3 16.4 20.7

[0108] fatty acid Transformation line (n=T1 allele number) g1CBEn g4CBEn g5CBEn 1 2 1 2 3 4 5 6 7 8 1 2 3 4 16:0 12.9 15.9 13.6 11.8 14.2 20.0 12.8 16.4 13.5 10.2 7.5 6.9 6.2 8.6 18:0 9.6 13.7 10.4 8.1 11.3 20.2 9.1 14.7 10.3 5.2 3.5 3.6 2.9 3.8 18:1 18.5 17.5 17.1 17.2 18.9 19.1 16.8 18.5 16.8 15.3 *36.5 *36.2 *48.9 *21.6 18:2 25.9 23.7 25.9 27.9 25.3 18.6 27.8 22.6 26.5 30.5 11.9 14.5 8.0 19.3 18:3 15.9 14.5 16.2 16.9 14.3 10.8 16.5 13.2 16.1 19.0 13.8 13.7 9.5 19.6 20:0 1.9 1.8 2.0 2.0 1.8 1.6 2.1 1.8 2.1 2.3 2.3 2.4 1.8 3.1 20:1 15.3 12.8 14.8 16.1 14.3 9.7 14.9 12.8 14.7 17.4 24.2 22.4 22.5 23.6

[0109] Subsequently, to determine whether cytosine base editing had indeed occurred in the targeted FAD2 region of the transgenic plants resulting in the observed increase, g5CBE1 and g5CBE3, which had the highest oleic acid content in T2 seeds, were isolated, and the next T2 generation was isolated.

[0111] 2. Analysis of base editing patterns and fat content of the FAD2 lineage

[0112] From the T1 lines of g5CBE1 and g5CBE3, seven (g5CBE11 to 17) and five (g5CBE31 to 35) T2 offspring were obtained and their FAD2 genotypes were analyzed. Genomic DNA was extracted from the leaves of each T2 plant, and Sanger sequencing was performed on the FAD2 gene in the CBE target region. Since C-to-G base substitutions were found at the C2 and C12 positions in g5CBE11 to 16, it is presumed that the protospacer adjacent motif (PAM) NGG was associated with positions 21-23, causing amino acid changes of A295G and D298E, respectively.

[0113] Among T2 plants, g5CBE12 and g5CBE14 showed distinct CG substitution chromatogram signals at corresponding positions, implying that (A295G; D298E)-type amino acid changes occurred. g5CBE11, g5CBE13, g5CBE15, and g5CBE16 showed mixed C-to-G and C-to-T signals at the C2 position, and the mixed signals of the original C and C-to-G at the C12 position suggest that, unless new base editing occurred in the T2 plants, the edited bi-alleles at the C2 position and the heterozygous C-to-G alleles at the C12 position from the T1 plants were transferred to their T2 plants.

[0114] In g5CBE17, only signals identical to the wild type were observed without specific signs of base editing. Among the g5CBE3 lines, the g5CBE31 plant also exhibited the A295G pattern resulting from a C-to-G conversion at the C2 position, accompanied by a C-to-T conversion at the C6 position, creating a synonymous mutation. In g5CBE32, an exclusive C-to-T substitution at the C2 position produced A295V, while C-to-T and C-to-G conversions at the C5 and C6 positions produced T296M. In the g5CBE33 plant, heterozygous or mosaic C-to-T substitutions were observed at the C2 position. Sequencing results for g5CBE34 and g5CBE35 show a mixed pool of sequencing traces, suggesting that genetic changes larger than point mutations, such as insertions and deletions, may have occurred (Figs. 5a, 6).

[0115] To investigate how lipid composition changed according to allele differences accompanied by amino acid changes, GC analysis was performed on T3 seeds obtained from each T2 plant. Oleic acid content increased to a maximum of 64.9%, 63.9%, and 69.2% in the T3 seeds of g5CBE12, g5CBE14, and g5CBE32, respectively, at the expense of polyunsaturated fatty acids, and two independent cytosine base-edited alleles simultaneously caused two amino acid changes at different residues (Fig. 5a).

[0116] The oleic acid content of T3 seeds of g5CBE11, g5CBE13, g5CBE15, and g5CBE16, in which bi-allelic edits at the C2 position and a heterozygous allele at the C12 position are expected to have separated, was found to be a moderate increase of 42.6% to 45.8% compared to the maximum increase in g5CBE12 and g5CBE14, implying that this is due to the C-to-T allele at the C2 position and / or the WT allele at the C12 position. g5CBE31 also showed an intermediate level of oleic acid increase, implying that A295G is responsible for partial inhibition of FAD2 activity. g5CBE33, possessing the heterozygous A295V, showed a small level of oleic acid increase of 25.7%, suggesting that A295V affects FAD2 activity in a dose-dependent manner (Fig. 5b). The oleic acid content of g5CBE34 and g5CBE35 showed a decent increase of 54% and 49.5%, respectively, but did not reach the maximum increase level as in g5CBE12 and g5CBE14.

[0118] 3. Isolation of the base-edited FAD2 allele

[0119] g5CBE11, g5CBE14, g5CBE31, g5CBE32, g5CBE33, and g5CBE34 were advanced to the next T3 generation. Since some of these were not yet genetically fixed in the T2 generation, homozygous alleles were isolated to clarify whether the change in fatty acid composition was due to possible T3 segregation (Figs. 5a and 5b; columns containing numbers marked with *). g5CBE11 was selected as the representative of the T2 group containing A295G / A295V bi-allelic due to the homozygous D298E mutation.

[0120] g5CBE14 and g5CBE32 correspond to the A295G; D298E and A295V; T296M allelic groups, respectively. g5CBE31 carries the A295G mutation and a closely related C-to-T mutation. g5CBE33 was selected as the heterozygous A295V allele. g5CBE34 was also included in the progression group, anticipating the segregation of the insertion-deletion allele in their T3 generation.

[0121] Sanger sequencing analysis was performed on the T3 offspring of each allelic group. Consequently, for each genotype, all possible base-edited T3 homozygous alleles g5CBE114 (A295G; D298E), g5CBE144 (A295G; D298E), g5CBE316 (A295G), g5CBE321 (A295V; T296M), and g5CBE337 (A295V) were isolated in this invention, and are hereinafter named FAD2-114, FAD2-144, FAD2-316, FAD2-321, and FAD2-337.

[0122] For the T3 offspring of g5CBE34, a 4bp insertion mutant allele was isolated and designated as FAD2-349, and it was expected that FAD2 would be knocked out due to an early stop codon. The isolated T3 offspring of g5CBE34 also exhibited a base-edited allele (A295G), indicating that the application of CBE induced both base substitution and insertion mutations in T2g5CBE34. GC analysis was performed on all T3 genotypes, and all homozygous base-edited alleles showed increased oleic acid content in them and in T4 seeds compared to wild-type Col-0.

[0123] The oleic acid content of FAD2-321 (A295V; T296M) was at most 64.7±5.7%, showing the highest increase in oleic acid among the base-edited FAD2 alleles, which is consistent with what was observed in their parental T2 generation (Fig. 5b).

[0124] This figure is higher than that of the previously reported FAD2-1 (A104T) (55.9±1.9%, Figure 3c, Supplementary Table S2). FAD2-114 and FAD2-144 (A295G; D298E) showed enhanced oleic acid production of 57.9±5.4% and 56.5±2.3%, respectively, which is higher than that of their isolated siblings (38±2.0%).

[0125] This implies that the increase above is due to simultaneous homozygous modifications occurring at two amino acids, which is segregated inheritance, and thus the previous generation g5CBE11 also showed a normal increase. In particular, the linoleic acid (18:2) and linolenic acid (18:3) content in FAD2-321 and FAD2-114 decreased drastically to 2.4% and 2% or less, respectively, while the linolenic acid (18:2) and linolenic acid (18:3) content in FAD2-1 was 2.4±0.2% and 6.2±0.5%, respectively, suggesting that the increase above is due to "weakened" desaturase activity of FA2 in their alleles. In particular, the oleic acid content in weakened alleles such as FAD2-321 was similar to the oleic acid content in the FAD2-349 knockout allele (59.5±10.4%) (Figs. 7a, 7c and Tables 3 to 5).

[0126] Fatty acid composition (%) of T3 seeds of g5CBE11n, g5CBE14n, g5CBE31n, g5CBE32n, g5CBE33n, and g5CBE34n (n=T3 allele number) fatty acid Col-0 FAD2-1 g5CBE11n 1 2 1 2 3 1 2 3 4 5 6 7 8 9 10 11 16:0 8.3 8.7 8.5 9.0 6.2 8.7 14.3 5.7 9.1 7.7 13.3 10.1 9.0 10.6 12.4 11.4 18:0 3.1 3.4 6.6 7.3 3.3 6.8 14.3 2.4 7.3 3.2 10.8 6.5 4.8 7.1 10.0 8.6 18:1 18.2 18.7 55.6 54.1 58.0 59.7 50.2 63.1 58.5 41.7 39.0 38.6 37.4 37.4 35.8 36.0 18:2 30.3 29.9 2.2 2.6 2.4 0.3 1.1 0.6 1.0 10.2 7.1 8.8 10.8 9.4 9.5 9.1 18:3 18.8 18.1 5.9 6.0 6.8 1.3 2.0 2.0 1.8 10.8 7.6 10.1 11.6 10.9 9.3 10.7 20:0 2.1 2.2 1.3 1.3 1.4 1.1 1.2 1.1 1.1 1.7 1.6 1.9 1.8 1.8 1.6 1.7 20:1 19.2 19.0 19.9 19.6 22.0 22.1 17.0 25.1 21.2 24.7 20.6 24.0 24.5 22.8 21.3 22.5

[0127] fatty acid g5CBE14n g5CBE31n g5CBE32n 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 16:0 8.6 8.7 11.0 9.5 10.8 10.5 12.3 13.2 10.8 13.9 7.8 7.5 8.8 6.6 13.7 5.1 5.7 5.2 18:0 6.8 6.5 9.8 7.8 9.2 8.8 10.5 11.6 8.0 12.4 3.6 3.4 7.5 4.3 14.3 2.0 2.3 2.1 18:1 59.4 58.1 55.1 58.2 53.3 55.1 34.2 32.0 33.5 33.0 37.4 37.7 61.0 64.4 55.4 69.7 67.2 70.6 18:2 0.4 0.3 0.4 0.4 0.8 0.4 12.1 12.6 13.6 12.3 14.3 13.6 0.3 0.3 0.6 0.6 1.8 0.4 18:3 1.8 1.5 1.5 1.5 2.5 1.5 11.2 11.5 13.1 10.5 13.9 14.3 1.3 1.6 1.2 1.7 2.4 1.6 20:0 1.2 1.1 1.1 1.1 1.1 1.0 1.7 1.8 1.8 1.7 1.9 1.8 1.1 1.2 0.8 0.9 1.0 0.9 20:1 21.8 23.9 21.2 21.7 22.4 22.8 17.9 17.4 19.3 16.2 21.1 21.7 20.0 21.6 13.9 19.9 19.7 19.2

[0128] fatty acid g5CBE33n g5CBE34n 1 2 3 4 5 6 7 1 2 3 4 5 6 7 8 9 10 11 15 16:0 11.6 9.2 9.8 11.3 9.9 9.9 11.2 11.0 9.9 8.4 14.6 9.6 11.0 11.3 11.3 5.4 6.2 6.5 8.9 18:0 7.9 4.6 5.2 7.5 4.9 4.4 8.2 9.6 8.1 6.0 15.1 7.3 8.9 10.2 7.2 2.3 3.1 2.9 3.2 18:1 21.2 21.5 21.8 21.8 16.4 16.0 30.6 43.2 45.6 45.4 52.1 42.9 42.7 44.2 28.9 66.9 50.9 50.3 30.3 18:2 22.6 25.2 24.6 23.7 29.3 31.0 16.8 8.0 7.9 8.5 0.5 9.0 8.8 6.8 18.2 0.5 7.8 8.4 18.7 18:3 15.1 16.8 16.6 15.0 18.1 17.8 12.8 8.8 8.7 10.5 1.0 10.9 10.2 9.0 15.5 1.8 9.5 9.6 17.0 20:0 2.4 2.3 2.4 2.2 2.4 2.4 1.8 1.4 1.3 1.4 1.0 1.4 1.3 1.3 1.8 1.1 1.5 1.4 1.8 20:1 19.0 20.4 19.7 18.4 19.2 18.5 18.7 17.9 18.5 19.7 15.7 18.8 17.0 17.2 17.1 22.0 21.0 20.9 20.1

[0129] 4. Characteristics of the growth response of the FAD2 allele

[0130] It was hypothesized that a specific "weakened" FAD2 allele of the present invention could maintain an increased oleic acid content while having severe growth defects such as those of the FAD2 knockout allele or weaker defects than the previously reported FAD2-1 allele.

[0131] In this regard, newly produced FAD2 alleles were identified according to phenotypic differences in terms of their growth responses. In this invention, the germination rate and root growth rate of each allele were analyzed under normal and stress conditions.

[0132] For experimental stress conditions for germination and root growth tests, 150 mM NaCl, 300 mM Mannitol, and 75 mM NaCl, 200 mM Mannitol were used in ½ MS media, respectively, simulating salt or water deficiency stress conditions.

[0133] To determine whether an increase in oleic acid content affects germination, germination tests were conducted for each allele by scoring seed germination at 12-hour intervals for the first 4 days after planting (Figs. 9a, 9b, and 9c). Under normal conditions, the germination frequencies of the tested alleles became similar after 4 days, showing a germination success rate of over 95%.

[0134] The FAD2-349, FAD2-321, and FAD2-144 alleles showed a slightly reduced germination rate (~72 hours; Fig. 9a), which is consistent with the result showing that these alleles have a relatively high oleic acid content compared to other alleles (Fig. 7c).

[0135] Under the two experimental stress conditions, the germination rates of FAD2-349 and FAD2-321 deteriorated sharply, along with FAD2-1, which exhibited significantly lower germination rates at all tested time points. However, FAD2-144, FAD2-316, and FAD2-337 maintained germination rates similar to the wild type. This suggests that FAD2 alleles with increased oleic acid content exhibited enhanced resistance to stress susceptibility during germination, in contrast to the cases of FAD2-1, FAD2-349, and FAD2-321. In particular, FAD2-144, which exhibited one of the highest levels of oleic acid content, contained two simultaneous amino acid changes (A295G; D295E, Fig. 7) and showed significant resistance to the stress conditions tested during germination compared to FAD2-1 (Figs. 9b and 9c).

[0136] Next, root growth under normal or stress conditions was evaluated by measuring the root length of 4-day-old seedlings from each FAD2 allele (Figs. 10d, 10e, and 10f). Under normal growth conditions, all FAD2 alleles tested showed reduced root length compared to wild-type Col-0. For example, FAD2-144 and FAD2-321 showed a significant reduction in root length, similar to that observed in FAD2-1 and knockout FAD2-349, suggesting that the corresponding allelic changes inhibited normal root development. On the other hand, FAD2-316 and FAD2-337 showed only minimal levels of root reduction. This implies that the allelic variations did not inhibit the normal growth process.

[0137] Stress treatment induced varying reductions in root length in seedlings of each tested allele (Figs. 10d and 10e). Under salt stress conditions, the knockout allele FAD2-349 showed a reduction in root length to 55.8% of that under normal conditions (Fig. 10f), which is consistent with the known growth defects of FAD2 knockout mutants under salt stress. In contrast, the root lengths of Col-0 and FAD2-1 were 66% and 63.6% of their normal growth lengths, respectively. Notably, FAD2-316 (A295G) and FAD2-337 (A295V) (Fig. 3), which exhibited an intermediate increase in oleic acid due to a single amino acid substitution, showed a less significant reduction in root length, at 82.9% and 79.7% of their normal lengths, respectively. This suggests that their growth is less affected by salt stress than that of the wild type. FAD2-144 (A295G; D298E) and FAD2-321 (A295V; T296M) have two amino acids simultaneously substituted and possess a higher oleic acid-producing allele (Fig. 7c), showing relative root growth of 67.8% and 69.2% under salt stress conditions, which is similar to the reduced levels observed in Col-0.

[0138] Growth defects were more clearly observed under water shortage stress conditions using 200 mM Mannitol, with the root length of FAD2-349 decreasing to 32.6%, while that of Col-0 decreased to 45.5%. FAD2-316 and FAD2-337 showed relative root lengths of 49.1% and 54%, respectively, indicating significantly increased resistance to water shortage stress compared to knockout FAD2-349 (32.6%) or the previously reported FAD2-1 (35.5%). The relative root lengths of FAD2-144 and FAD2-321 were 40.4% and 41.1%, respectively, suggesting that while their sensitivity to stress conditions increased compared to Col-0, they could tolerate water shortage stress better than FAD2-349 and FAD2-1.

Claims

Claim 1 A plant that expresses a FAD2 protein, wherein the protein is composed of a sequence corresponding to the sequence of SEQ ID NO. 1, and the amino acid at the position corresponding to the 295th position in SEQ ID NO. 1 is Gly, and wherein the plant produces seeds with an increased oleic acid content compared to wild-type seeds. Claim 2 In claim 1, the protein is a plant in which the amino acid at the position corresponding to the 298th position in sequence number 1 is Glu. Claim 3 delete Claim 4 In claim 1, the plant body is a plant body that is sesame, sesame, perilla, peanut, rice bran, soybean, rapeseed, flax, castor oil plant, palm tree, olive, camellia, safflower, hemp, flax, paulownia, sunflower, olive, peanut, camellia, castor oil plant, rapeseed, Arabidopsis thaliana, hazelnut tree, red eucalyptus, rice, resquerella, castor oil plant, or corn. Claim 5 A seed produced by the plant of claim 1, wherein the seed has an increased oleic acid content compared to the wild type. Claim 6 A method for producing a plant, comprising the step of modifying a gene encoding a FAD2 protein having a sequence corresponding to the sequence of SEQ ID NO. 1 in a plant so that the amino acid at the position corresponding to the 295th position in SEQ ID NO. 1 is substituted with Gly to code for a protein, wherein the plant produces seeds having an increased oleic acid content compared to wild-type seeds. Claim 7 A method for producing a plant according to claim 6, further comprising the step of selecting a plant that expresses a Gluin protein, wherein the amino acid at the position corresponding to the 298th position in sequence number 1 is among the mutated plant. Claim 8 delete Claim 9 A method for producing a plant in claim 6, wherein the substitution is performed using gRNA of SEQ ID NO. 2.

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