WO2022139463A1 - Solanum lycopersicum-derived apx4 gene to control ascorbic acid content of tomato fruits and use thereof - Google Patents

Solanum lycopersicum-derived apx4 gene to control ascorbic acid content of tomato fruits and use thereof Download PDF

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WO2022139463A1
WO2022139463A1 PCT/KR2021/019619 KR2021019619W WO2022139463A1 WO 2022139463 A1 WO2022139463 A1 WO 2022139463A1 KR 2021019619 W KR2021019619 W KR 2021019619W WO 2022139463 A1 WO2022139463 A1 WO 2022139463A1
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tomato
gene
ascorbic acid
apx4
derived
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PCT/KR2021/019619
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French (fr)
Korean (ko)
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정영희
심재성
도주희
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전남대학교산학협력단
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Priority claimed from KR1020200181817A external-priority patent/KR102264215B1/en
Priority claimed from KR1020210073635A external-priority patent/KR102280955B1/en
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    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01HNEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/06Processes for producing mutations, e.g. treatment with chemicals or with radiation
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01HNEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/10Seeds
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01HNEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/82Solanaceae, e.g. pepper, tobacco, potato, tomato or eggplant
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/11DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • CCHEMISTRY; METALLURGY
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    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004Oxidoreductases (1.)
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14Hydrolases (3)
    • C12N9/16Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22Ribonucleases RNAses, DNAses

Definitions

  • the present invention relates to a tomato-derived APX4 (Ascorbate Peroxidase 4) gene that regulates the content of ascorbic acid in tomato fruits and uses thereof.
  • tomato-derived APX4 Ascorbate Peroxidase 4
  • Tomato Solanum lycopersicum
  • Solanum lycopersicum is an economical horticultural crop widely cultivated worldwide, and contains a lot of dietary fiber and functional substances, so research for developing high-functional economical varieties is being actively conducted.
  • antioxidants and carotenoids such as lycopene and potassium, it is also in the spotlight as a material for health functional foods.
  • Ascorbic acid is a substance acting as vitamin C and is a soluble redox molecule that regulates reactive oxygen specis. Ascorbic acid must be absorbed from the substance. Ascorbic acid is found mainly in plants and fruits. The amount of ascorbic acid varies greatly depending on the plant species, and it is known that tomatoes contain relatively low ascorbic acid (10-40 mg/100g fw) compared to other fruits. Therefore, the nutritional and industrial utilization of tomatoes can be greatly improved through the development of tomatoes with increased ascorbic acid content.
  • the CRISPR/Cas9 system since the CRISPR/Cas9 system has very high target specificity and can recognize methylated DNA as a target, it can be applied to a wide variety of genes. There is this.
  • the CRISPR/Cas9 system since it requires only Cas9 protein and sgRNA (single guide RNA), it has the advantage of being able to introduce target traits at a lower cost compared to the above two methods.
  • Korea Patent Application Publication No. 2019-0043841 discloses 'a method of reducing ethylene production by LeMADS-RIN gene editing in a plant using the CRISPR/Cas9 system', and in Korea Patent No. 2090157, ' Although the wild rice-derived APX9 gene for controlling plant height, seed size and seeding season and its use' has been disclosed, 'a tomato-derived APX4 gene for regulating the content of ascorbic acid in tomato fruit and its use' of the present invention is not described.
  • the present invention was derived from the above needs, and the present inventors isolated SlAPX4 ( Solanum lycopersicum Ascorbate Peroxidase ) genes present in tomato, SlAPX4 specifically expressed in the fruit development stage, and SlAPX6 gene, which is highly expressed in leaves . Afterwards, VIGS (Virus-Induced Gene Silencing) vectors that can act in three regions were prepared for SlAPX4 and SlAPX6 , respectively, and the prepared vector was introduced into immature green tomato fruits through Agrobacterium, followed by red-ripeing. The content of ascorbic acid in the step was analyzed.
  • SlAPX4 Solanum lycopersicum Ascorbate Peroxidase
  • VIGS Virus-Induced Gene Silencing
  • the present inventors developed a transgenic tomato by constructing a CRISPR/Cas9 vector targeting SlAPX4 , and by analyzing the nucleotide sequence of the target site of SlAPX4 in the transgenic tomato, it was confirmed that editing occurred on the tomato SlAPX4 gene sequence, As a result of comparing the ascorbic acid content in the wild-type tomato and the gene-modified tomato of the Ripen Red period, which is a reproductive period, the present invention completed.
  • the present invention comprises the step of regulating the expression of APX4 (Ascorbate Peroxidase 4) protein coding gene derived from tomato ( Solanum lycopersicum ) consisting of the amino acid sequence of SEQ ID NO: 2 Controlling the content of ascorbic acid in tomato fruits provide a way
  • the present invention comprises the steps of (a) transforming tomato plant cells with a recombinant vector containing a tomato-derived APX4 protein coding gene; and (b) re-differentiating the transformed tomato plant from the transformed tomato plant cells; provides a method for producing a transformed tomato plant in which the ascorbic acid content of the fruit is controlled, including.
  • the present invention provides a transgenic tomato plant having an increased content of ascorbic acid in the fruit produced by the above method and a transformed seed thereof.
  • the present invention provides a composition for regulating the content of ascorbic acid in tomato fruits, which contains a tomato-derived APX4 protein encoding gene consisting of the amino acid sequence of SEQ ID NO: 2 as an active ingredient.
  • the present invention consisting of the nucleotide sequence of SEQ ID NO: 30, DNA encoding a guide RNA (guide RNA) specific to the target nucleotide sequence of the tomato-derived APX4 gene and a nucleic acid sequence encoding an endonuclease protein a recombinant vector comprising; or a complex (ribonucleoprotein) of a guide RNA and an endonuclease protein specific for the target nucleotide sequence of the tomato-derived APX4 gene consisting of the nucleotide sequence of SEQ ID NO: 30; as an active ingredient, increase the ascorbic acid content of tomato fruits It provides a composition for genome editing for
  • the present invention provides a composition for genome editing for increasing the content of ascorbic acid in tomato fruits, comprising, as an active ingredient, a guide RNA specific for the target nucleotide sequence of the tomato-derived APX4 gene, consisting of the nucleotide sequence of SEQ ID NO: 30 do.
  • the present invention comprises the steps of (a) introducing a guide RNA and an endonuclease protein specific for the target nucleotide sequence of the tomato-derived APX4 gene consisting of the nucleotide sequence of SEQ ID NO: 30 into tomato plant cells to correct the genome; And (b) the step of redifferentiating the tomato plant from the tomato plant cells in which the genome has been corrected; provides a method for producing a genome-edited tomato plant comprising, the ascorbic acid content of the fruit is increased.
  • the present invention provides a genome-corrected tomato plant having an increased content of ascorbic acid in the fruit produced by the above method, and a seed whose genome has been corrected.
  • the technique of increasing the ascorbic acid content in tomato fruits through SlAPX4 gene expression silencing presented in the present invention increases the accumulation of ascorbic acid by inhibiting the decomposition of ascorbic acid in the plant. There will be.
  • the APX4 gene editing technology presented in the present invention induces mutations that cannot be distinguished from natural mutations, it is costly, unlike Genetically Modified Organism (GMO) crops, which consume enormous costs and time to evaluate safety and environmental hazards. and time is expected to be saved.
  • GMO Genetically Modified Organism
  • 1 is a diagram showing the coding sequence of SlAPX4 (A) and SlAPX6 (B) present in tomato.
  • FIG. 2 is a graph analyzing the expression patterns of SlAPX4 and SlAPX6 at each stage of tomato fruit development in Heinz ( Solanum lycopersicum cv. Heinz) and S. lycopersicum cv. Micro-Tom tomatoes.
  • FIG. 3 is a diagram schematically illustrating the structure of the VIGS vector constructed to suppress the expression of SlAPX4 and SlAPX6 .
  • FIG. 5 shows the location of the target sequence in the SlAPX4 gene (A) and the construction of a recombinant vector (B) carrying the Cas9 protein and sgRNA targeting the SlAPX4 gene.
  • Figure 6 is the result of confirming the target sequence change (mutation) in the second generation of transgenic plants to determine whether the generation of the developed transgenic plant trait is fixed (A) and the ascorbic acid content of the second generation of SlAPX4 gene-corrected plants. It is a graph (B).
  • the present invention provides a method for regulating the content of ascorbic acid in tomato fruit, comprising the step of regulating the expression of APX4 (Ascorbate Peroxidase 4) protein-coding gene derived from tomato ( Solanum lycopersicum ) consisting of the amino acid sequence of SEQ ID NO: 2.
  • APX4 Ascorbate Peroxidase 4
  • the APX4 protein may preferably consist of the amino acid sequence of SEQ ID NO: 2, more preferably, the 167th to 250th amino acid sequence among the amino acid sequence of SEQ ID NO: 2 may be made, but is not limited thereto.
  • the 167th to 250th amino acid sequence of the amino acid sequence of SEQ ID NO: 2 corresponds to the 499th to 753th nucleotide sequence in the nucleotide sequence of SEQ ID NO: 1.
  • the scope of the tomato-derived APX4 protein according to the present invention includes a protein having the amino acid sequence shown in SEQ ID NO: 2 and functional equivalents of the protein.
  • the term "functional equivalent” means at least 70% or more, preferably 80% or more, more preferably 90% of the amino acid sequence represented by SEQ ID NO: 2 as a result of addition, substitution or deletion of amino acids. As described above, more preferably, it refers to a protein having a sequence homology of 95% or more and exhibiting physiological activity substantially identical to that of the protein represented by SEQ ID NO: 2.
  • “Substantially homogenous physiological activity” means the activity of regulating the content of ascorbic acid in tomato fruit.
  • the method for regulating the ascorbic acid content of tomato fruits according to the present invention may be to increase the ascorbic acid content of fruits compared to non-transformed plants by inhibiting the expression of the tomato-derived APX4 protein coding gene in plant cells, but this not limited
  • the inhibition of expression of the gene encoding the tomato-derived APX4 protein is a virus-induced gene silencing (VIGS) system, RNAi or antisense RNA, T-DNA insertion, endogenous to the tomato-derived APX4 protein-coding gene.
  • VIPGS virus-induced gene silencing
  • RNAi or antisense RNA RNAi or antisense RNA
  • T-DNA insertion endogenous to the tomato-derived APX4 protein-coding gene.
  • Transposon, mutagenesis through X-ray or ⁇ -ray irradiation, or inhibiting (suppressing) the expression of the tomato-derived APX4 protein-coding gene using a CRISPR/Cas9 gene correction system, etc. but is not limited thereto
  • any conventional method in the art for inhibiting gene expression may be possible.
  • the present invention provides a gene encoding the tomato-derived APX4 protein.
  • the gene encoding the tomato-derived APX4 protein includes both genomic DNA and cDNA.
  • the gene encoding the tomato-derived APX4 protein of the present invention may include the nucleotide sequence of SEQ ID NO: 1.
  • homologs of the nucleotide sequence are included within the scope of the present invention.
  • the gene comprises a nucleotide sequence having at least 70%, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% sequence homology to the nucleotide sequence of SEQ ID NO: 1, respectively.
  • the "% sequence homology" for a polynucleotide is determined by comparing two optimally aligned sequences with a comparison region, wherein a portion of the polynucleotide sequence in the comparison region is a reference sequence (additions or deletions) to the optimal alignment of the two sequences. may include additions or deletions (ie, gaps) compared to (not including).
  • the term “recombinant” refers to a cell in which the cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a peptide, a heterologous peptide or a protein encoded by the heterologous nucleic acid.
  • Recombinant cells can express genes or gene segments not found in the native form of the cell, either in sense or antisense form.
  • Recombinant cells can also express genes found in cells in a natural state, but the genes are modified and re-introduced into cells by artificial means.
  • vector is used to refer to a DNA fragment(s), a nucleic acid molecule, that is delivered into a cell.
  • the vector replicates DNA and can be reproduced independently in a host cell.
  • carrier is often used interchangeably with “vector.”
  • expression vector refers to a recombinant DNA molecule comprising a desired coding sequence and a suitable nucleic acid sequence necessary for expression of an operably linked coding sequence in a particular host organism.
  • the vector of the present invention can typically be constructed as a vector for cloning or expression.
  • the vector of the present invention can be constructed using a prokaryotic cell or a eukaryotic cell as a host.
  • a prokaryotic cell when the vector of the present invention is an expression vector and a prokaryotic cell is used as a host, a strong promoter capable of propagating transcription (eg, pL ⁇ promoter, Trp promoter, Lac promoter, T7 promoter, Tac promoter, etc.); It generally includes a ribosome binding site for initiation of translation and a transcription/translation termination sequence.
  • pL ⁇ promoter e.g, pL ⁇ promoter, Trp promoter, Lac promoter, T7 promoter, Tac promoter, etc.
  • Escherichia coli When Escherichia coli is used as the host cell, the promoter and operator sites of the E. coli tryptophan biosynthesis pathway, and the left-handed promoter (pL
  • the promoter is a promoter suitable for transformation, preferably CaMV 35S promoter, actin promoter, ubiquitin promoter, pEMU promoter, MAS promoter or histone promoter, preferably CaMV 35S promoter.
  • a promoter suitable for transformation preferably CaMV 35S promoter, actin promoter, ubiquitin promoter, pEMU promoter, MAS promoter or histone promoter, preferably CaMV 35S promoter.
  • promoter refers to a region upstream of DNA from a structural gene and refers to a DNA molecule to which RNA polymerase binds to initiate transcription.
  • a “plant promoter” is a promoter capable of initiating transcription in a plant cell.
  • a “constitutive promoter” is a promoter that is active under most environmental conditions and developmental states or cell differentiation. A constitutive promoter may be preferred in the present invention since the selection of transformants can be made by various tissues at various stages. Thus, constitutive promoters do not limit the selectivity.
  • the recombinant vector of the present invention can be constructed by methods well known to those skilled in the art.
  • the method includes in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology.
  • the DNA sequence can be effectively linked to a suitable promoter in an expression vector to direct mRNA synthesis.
  • the vector may also include a ribosome binding site as a translation initiation site and a transcription terminator.
  • a preferred example of the recombinant vector is a VIGS vector or an RNAi vector.
  • VIGS refers to a phenomenon in which expression of an endogenous gene of the introduced gene is suppressed when a plant is infected after introducing a plant gene into a viral vector. It is a type of post-transcriptional gene silencing (PTGS), and has characteristics of post-transcriptional, RNA turnover, and nucleotide sequence-specific.
  • the VIGS vector can be used as a transient expression vector that can be temporarily expressed in a plant into which a foreign gene is introduced, and a plant expression vector that can be permanently expressed in a plant into which the foreign gene is introduced.
  • a preferred example of a plant expression vector is a Ti-plasmid vector capable of transferring a part of itself, the so-called T-region, into a plant cell when present in a suitable host such as Agrobacterium tumefaciens .
  • a Ti-plasmid vector capable of transferring hybrid DNA sequences into plant cells, or protoplasts from which new plants can be produced that properly insert the hybrid DNA into the genome of the plant and have.
  • a particularly preferred form of the Ti-plasmid vector is the so-called binary vector as claimed in EP 0 120 516 B1 and US Pat. No. 4,940,838.
  • viral vectors such as those that can be derived from double-stranded plant viruses (eg CaMV) and single-stranded viruses, gemini viruses, etc.
  • CaMV double-stranded plant viruses
  • gemini viruses single-stranded viruses
  • it may be selected from an incomplete plant viral vector.
  • the use of such vectors can be advantageous, especially when it is difficult to adequately transform a plant host.
  • the recombinant expression vector may preferably comprise one or more selectable markers.
  • the marker is a nucleic acid sequence having a characteristic that can be selected by a conventional chemical method, and includes all genes capable of distinguishing a transformed cell from a non-transformed cell.
  • the marker gene may be an antibiotic resistance gene (dominant drug resistance gene), but is not limited thereto.
  • any host cell known in the art including microalgae and microorganisms, may be used, for example, E. coli JM109, E. coli BL21, Bacillus genus strains such as E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis , B.
  • Salmonella typhimurium Salmonella typhimurium
  • Serratia marcescens Serratia marcescens
  • various Pseudomonas enterobacteriaceae and strains such as (Pseudomonas) species.
  • yeast eg, Saccharomyce cerevisiae
  • insect cells eg, human cells
  • human cells eg, CHO cell line (Chinese hamster ovary), W138, BHK, COS-7) , 293, HepG2, 3T3, RIN and MDCK cell lines
  • plant cells preferably plant cells.
  • Transformation of a plant refers to any method of transferring DNA into a plant. Such transformation methods need not necessarily have a period of regeneration and/or tissue culture. Transformation of plant species is now common for plant species including both monocots as well as dicots. In principle, any transformation method can be used to introduce the hybrid DNA according to the invention into suitable progenitor cells. Methods include the calcium/polyethylene glycol method for protoplasts (Krens, F.A. et al., 1982, Nature 296, 72-74; Negrutiu I. et al., 1987, Plant Mol. Biol. 8, 363-373), protoplasts. Electroporation (Shillito R.D. et al., 1985 Bio/Technol.
  • a preferred method according to the present invention comprises Agrobacterium mediated DNA delivery.
  • the present invention also relates to the present invention.
  • the tomato-derived APX4 protein may include a protein consisting of the amino acid sequence of SEQ ID NO: 2 and a functional equivalent of the protein, and the specific details are as described above same.
  • the method for transforming the plant cell is as described above, and the method for redifferentiating the transgenic plant from the transformed plant cell is known in the art. Any method may be used.
  • Transformed plant cells must be redifferentiated into whole plants. Techniques for the redifferentiation of mature plants from callus or protoplast cultures are well known in the art for a number of different species.
  • Plant cell used for transformation of a plant may be any plant cell.
  • Plant cells are cultured cells, cultured tissues, cultured organs or whole plants.
  • Plant tissue refers to differentiated or undifferentiated plant tissues, such as, but not limited to, roots, stems, leaves, pollen, seeds, cancer tissues and various types of cells used in culture, ie, single cells, protoplasts. (protoplast), shoots and callus tissue.
  • the plant tissue may be in planta or in an organ culture, tissue culture or cell culture state.
  • the non-transformed plant that is, the fruit of the tomato plant compared to the wild-type tomato My ascorbic acid content may be increased, but is not limited thereto.
  • the tomato-derived APX4 protein coding Inhibition of gene expression may be to inhibit the expression of APX4 protein-coding gene by transforming tomato plant cells with a recombinant vector containing sense or antisense DNA, or microRNA for the tomato-derived APX4 gene. It is not limited thereto, and a gene expression inhibition technique known in the art may be used.
  • the present invention also provides a transgenic tomato plant having an increased content of ascorbic acid in the fruit produced by the method for producing the transgenic tomato plant, and a transformed seed thereof.
  • the transgenic tomato plant with an increased content of ascorbic acid in the fruit according to the present invention is characterized in that the content of ascorbic acid in the fruit is increased by inhibiting the expression of the APX4 protein coding gene.
  • the present invention also provides a composition for regulating the content of ascorbic acid in tomato fruits, which contains a tomato-derived APX4 protein encoding gene consisting of the amino acid sequence of SEQ ID NO: 2 as an active ingredient.
  • the composition for controlling the ascorbic acid content of tomato fruits of the present invention includes a tomato-derived APX4 protein coding gene capable of controlling the ascorbic acid content of tomato fruits as an active ingredient, and when the expression of the gene is inhibited, the ascorbic acid content of tomato fruits is reduced can increase
  • the present invention also provides a nucleic acid sequence encoding a guide RNA (guide RNA) specific to the target nucleotide sequence of the tomato-derived APX4 gene, and a nucleic acid sequence encoding an endonuclease protein, consisting of the nucleotide sequence of SEQ ID NO: 30 a recombinant vector comprising; or a complex (ribonucleoprotein) of a guide RNA and an endonuclease protein specific for the target nucleotide sequence of the tomato-derived APX4 gene consisting of the nucleotide sequence of SEQ ID NO: 30; as an active ingredient, increase the ascorbic acid content of tomato fruits It provides a composition for genome editing for
  • the term “genome/gene editing” refers to a technology capable of introducing a target-directed mutation into the genome sequence of animal and plant cells, including human cells, and one or more nucleic acid molecules by DNA cleavage. Knock-out or knock-in a specific gene by deletion, insertion, substitution, etc. of coding) refers to a technology that can introduce mutations into DNA sequences.
  • the genome editing may be, in particular, introducing a mutation into a plant using an endonuclease, such as a Cas9 (CRISPR associated protein 9) protein and a guide RNA.
  • 'gene editing' may be used interchangeably with 'gene editing'.
  • target gene refers to some DNA in the genome of a plant to be corrected through the present invention, is not limited to the type of the gene, and may include both a coding region and a non-coding region. A person skilled in the art can select the target gene according to the desired mutation for the genome-corrected plant to be prepared, depending on the purpose.
  • guide RNA is a short single-stranded RNA, and refers to an RNA specific for a target DNA among nucleotide sequences encoding a target gene, and all or part of the target DNA nucleotide sequence and all or part of the nucleotide sequence are complementary Thus, it refers to a ribonucleic acid that leads the endonuclease protein to the corresponding target DNA sequence.
  • the guide RNA may be a dual RNA comprising two RNAs, ie, crRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA); or a single chain comprising a first site comprising a sequence that is all or partly complementary to a nucleotide sequence in a target gene and a second site comprising a sequence that interacts with an endonuclease (especially an RNA-guided nuclease)
  • sgRNA single guide RNA
  • the endonuclease has activity in the target nucleotide sequence, it may be included in the scope of the present invention without limitation, and the type or endonuclease used together It can be prepared and used according to a technique known in the art in consideration of the microorganisms derived from the nuclease and the like.
  • the guide RNA may be transcribed from a plasmid template, transcribed in vitro (eg, oligonucleotide double-stranded), or synthesized guide RNA, but is not limited thereto.
  • the endonuclease protein is Cas9, Cpf1 (also known as Cas12a), TALEN (Transcription activator-like effector nuclease), ZFN (Zinc Finger Nuclease) or a functional analog thereof. It may be at least one selected from the group consisting of, preferably, an RNA-guided nuclease, such as Cas9 or Cpf1, and more preferably, a Cas9 protein, but is not limited thereto.
  • the Cas9 protein is a Cas9 protein derived from Streptococcus pyogenes , a Cas9 protein derived from Campylobacter jejuni , S. thermophilus or Streptococcus aureus ( S. aureus )-derived Cas9 protein, Neisseria meningitidis -derived Cas9 protein, Pasteurella multocida -derived Cas9 protein, Francisella novicida ) derived Cas9 protein, etc. It may be one or more selected from the group consisting of, but is not limited thereto.
  • Cas9 protein or genetic information thereof can be obtained from a known database such as GenBank of the National Center for Biotechnology Information (NCBI). As the Cas9 gene information, a known sequence may be used as it is, or a sequence optimized for the codon of the subject (organism) to be transduced may be used, but is not limited thereto.
  • Cas9 protein is an RNA-guided DNA endonuclease enzyme that induces double-stranded DNA breaks.
  • PAM Protospacer Adjacent Motif
  • the guide RNA and the endonuclease protein form a ribonucleoprotein complex to operate as RNA-Guided Engineered Nuclease (RGEN).
  • RGEN RNA-Guided Engineered Nuclease
  • the tomato-derived APX4 gene may consist of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 38, but is not limited thereto.
  • the CRISPR/Cas9 system used introduces a double helix break at a specific position of a specific gene to be corrected, and an insertion-deletion caused by incomplete repair induced in the DNA repair process.
  • InDel is a gene editing method by NHEJ (non-homologous end joining) mechanism that induces mutations.
  • the present invention provides a genome editing composition for increasing the content of ascorbic acid in tomato fruits, comprising, as an active ingredient, a guide RNA specific for the target nucleotide sequence of the tomato-derived APX4 gene, consisting of the nucleotide sequence of SEQ ID NO: 30 do.
  • the guide RNA of the present invention may preferably be in the form of a single guide RNA (sgRNA), but is not limited thereto, and the sgRNA is specific for an endonuclease binding sequence in addition to a guide RNA specific for a target sequence It may include RNA, and preferably, the guide RNA may consist of the nucleotide sequence of SEQ ID NO: 37, but is not limited thereto.
  • sgRNA single guide RNA
  • the present invention also relates to the present invention.
  • the introduction of the guide RNA and endonuclease protein of step (a) into tomato plant cells comprises the nucleotide sequence of SEQ ID NO: 30, tomato-derived APX4 gene a recombinant vector comprising DNA encoding a guide RNA specific for a target nucleotide sequence and a nucleic acid sequence encoding an endonuclease protein;
  • a complex (ribonucleoprotein) of a guide RNA and an endonuclease protein specific to the target nucleotide sequence of the tomato-derived APX4 gene consisting of the nucleotide sequence of SEQ ID NO: 30 may be used, but is not limited thereto.
  • the nucleotide sequence of SEQ ID NO: 30 is compared to the nucleotide sequence of SEQ ID NO: 27 to SEQ ID NO: 29, which are other guide RNA target sequences, at the target site of the APX4 gene. It is characterized by high InDel mutagenesis efficiency.
  • the method for transducing the complex of the guide RNA and the endonuclease protein into plant cells is the same as described above.
  • introducing a recombinant vector including a DNA encoding a guide RNA specific for the target nucleotide sequence and a nucleic acid sequence encoding an endonuclease protein into a plant cell means a transformation method. Transformation of plant species is now common for plant species including both monocots as well as dicots. In principle, any transformation method can be used to introduce the recombinant vector according to the invention into suitable progenitor cells.
  • the recombinant vector when the recombinant vector is transformed into a plant cell, it binds to an endonuclease protein having DNA binding and cleavage activity and the endonuclease protein and the sgRNA leading to the endonuclease protein as the target sequence is expressed together.
  • plant cells into which the guide RNA and endonuclease protein specific to the target nucleotide sequence are introduced are the same as described above.
  • the present invention also provides a genome-corrected tomato plant having an increased content of ascorbic acid in the fruit produced by the method for producing the genome-corrected tomato plant, and a seed whose genome is corrected.
  • the genome-corrected tomato plant with an increased content of ascorbic acid in the fruit according to the present invention was obtained by correcting the APX4 (Ascorbate Peroxidase 4) gene specifically expressed in the tomato fruit using the CRISPR/Cas9 system, and the tomato-derived APX4 gene was -Out, it is a genome-corrected tomato plant having a trait that the ascorbic acid content of the fruit is increased compared to a tomato plant without genome editing.
  • APX4 Ascorbate Peroxidase 4
  • tomato 'micro-tome' Solanum lycopersicum cv. Micro-Tom
  • Tomato seeds were germinated in a medium composed of MS salt 4.4 g/L, sucrose 30 g/L, and plant agar 8 g/L, 22 ⁇ 24°C, 16 hours of light, 8 hours of dark. It was grown in a culture room maintained in a condition.
  • Realtime PCR analysis was performed after adding 4 ⁇ l of cDNA diluted 1/10, 3 ⁇ l of sterile distilled water, 2 ⁇ l of forward primer, 2 ⁇ l of reverse primer, and 10 ⁇ l of SYBR Green (Qiagen, Germany) at 95°C for 15 minutes, 95°C 50 cycles were performed at 20 sec, 55 °C 30 sec, and 72 °C 30 sec, and the fluorescence signal was detected at every cycle.
  • Primer information used for gene expression analysis is shown in Table 1.
  • TRV2(pYL156) was inoculated with 5 ⁇ l of E.coli stock in LB broth containing 50 mg/L of kanamycin (LB broth High Salt 25 g/L) and cultured for one day at 37°C, followed by Nucleospin plasmid easypure kit ( The plasmid was extracted through MACHEREY-NAGEL, Germany). The extracted plasmid was treated with EcoR I and BamHI and reacted for one day at 37° C.
  • TRV2 TRV2 (pYL156) vector 4 ⁇ l, insert 4 ⁇ l, T4 ligase (cosmogentech), 10X buffer (cosmogentech) 1 ⁇ l, 2 ⁇ l of sterile distilled water was added, and ligation was performed at room temperature for 2 minutes and 30 seconds and on ice for 10 minutes to proceed with ligation.
  • Fig. 3 The constructed recombinant vector was inserted into Agrobacterium tumefaciens GV3101. The presence or absence of a gene was confirmed through PCR and sequencing, and information on primers used for insert synthesis and PCR analysis is described in Table 1 above.
  • the strain was resuspended by adding 10 mM MgCl 2 at a ratio of 1:1 with the medium in which the strain was passaged, and then MgCl 2 was added to adjust the OD to 1.0 at 600 .
  • MgCl 2 was added to adjust the OD to 1.0 at 600 .
  • dark culture was performed at room temperature for 4 hours. After that, the fungus was injected into the fruits of the tomato mature green stage.
  • sgRNA to which the CRISPR/Cas9 system can be applied to the SlAPX4 gene sequence was prepared and the vector (pAGM4723::NPTII::Cas9::sgRNA1-4 using the Golden Gate system) ::L3E) was prepared and then inserted into Agrobacterium tumefaciens LBA4404. The presence or absence of the gene was confirmed through PCR and nucleotide sequence analysis using the primers shown in Table 4.
  • the culture solution was divided into two and co-cultured with the cotyledon leaf pieces of the cut tomato for 10 minutes, and then the cotyledon leaves were arranged in MT-COM (co-culture medium) so that the pores of the leaves face down, and the culture vessel was It was sealed and darkly cultured for 2 days.
  • tomato cotyledon pieces in MT-COM medium were transferred to MT-SRM (shoot induction medium) with the stomata of the leaves facing upward.
  • MT-SEM shoot elongation medium
  • Tissue culture medium composition co-culture medium Co-culture medium
  • New colostrum induction medium (Shoot) induction medium)
  • New growth medium (Shoot) elongation medium)
  • Root induction medium Root induction medium (Root) induction medium) 15 g/L sucrose 30 g/L sucrose 15 g/L sucrose 15 g/L sucrose 4.4 g/L MS salt 4.4 g/L MS salt 4.4 g/L MS salt 4.4 g/L MS salt 4.4 g/L MS salt 1 ⁇ M IAA 50 mg/L kanamycin 50 mg/L kanamycin 50 mg/L kanamycin 10 ⁇ M Zeatin 0.1mg/L IAA 250 mg/L Cefotaxim 2 mg/L IBA 200 ⁇ M acetosyringone 2 mg/L Zeatin 8 g/L plant agar 250 mg/L Cefotaxim 8 g/L plant agar 250 mg/L Cefotaxtim 8 g/
  • PCR primer information for transformant confirmation and target site mutation confirmation is shown in Table 4. Thereafter, the change in the nucleotide sequence of the target site was confirmed through sequencing, and it was determined whether the target site was mutated by the CRISPR/Cas9 system.
  • Ascorbic acid was measured in the fruit using Abcam's Ascorbic Acid Assay Kit. Wild-type and ripen red fruits of plants inducing gene silencing through the VIGS system or transgenic plants by the CRISPR/Cas 9 system were used for the experiment. After washing 40 mg of fruit tissue with PBS solution, put it in a tube with 400 ⁇ l of sterile distilled water and homogenize it using a homogenizer. After centrifuging the sample at 4° C. at 13,000 rpm for 5 minutes, take the supernatant and take the supernatant according to the manufacturer’s analysis method. The ascorbic acid content of each sample was measured at OD 593 using a spectrophotometer (microplate reader).
  • SlAPX4 showed a higher expression level in fruit tissues than SlAPX6 (FIG. 2C). From the above results, it was confirmed that SlAPX4 is a gene whose expression is increased in the late stage of fruit development, while SlAPX6 is a gene that is highly expressed in leaves and low in expression in the stage of fruit development.

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Abstract

The present invention relates to a method for controlling the ascorbic acid content of tomato fruits, the method comprising a step of regulating the expression of a gene encoding Solanum lycopersicum-derived ascorbate peroxidase 4 (APX4) protein.

Description

토마토 열매의 아스코르브산 함량을 조절하는 토마토 유래 APX4 유전자 및 이의 용도Tomato-derived APX4 gene regulating ascorbic acid content in tomato fruit and use thereof
본 발명은 토마토 열매의 아스코르브산 함량을 조절하는 토마토 유래 APX4 (Ascorbate Peroxidase 4) 유전자 및 이의 용도에 관한 것이다.The present invention relates to a tomato-derived APX4 (Ascorbate Peroxidase 4) gene that regulates the content of ascorbic acid in tomato fruits and uses thereof.
본 결과물은 농촌진흥청의 차세대농작물신육종기술개발(R&D)사업의 지원을 받아 연구되었습니다(PJ014874). This result was researched with support from the R&D project of the Rural Development Administration (PJ014874).
토마토(Solanum lycopersicum)는 전 세계적으로 널리 재배되는 경제성 원예작물로 식이섬유질 및 기능성 물질을 많이 포함하고 있어 고기능성 경제성 품종 개발을 위한 연구가 활발히 진행되고 있다. 특히 항산화 물질 및 카로티노이드계의 리코펜(lycopene), 칼륨 등을 함유하고 있어 건강기능식품의 재료로도 각광받고 있다.Tomato ( Solanum lycopersicum ) is an economical horticultural crop widely cultivated worldwide, and contains a lot of dietary fiber and functional substances, so research for developing high-functional economical varieties is being actively conducted. In particular, as it contains antioxidants and carotenoids such as lycopene and potassium, it is also in the spotlight as a material for health functional foods.
아스코르브산은 비타민 C로 작용하는 물질로서 활성화산소종(Reactive oxygen specis)을 조절하는 용해성 산화환원분자로, 인간의 경우 아스코르브산을 합성하는 guluno-1,4γ-lactone oxidase (GuLo)가 결핍되어 있어 외부 물질로부터 아스코르브산을 흡수해야 한다. 아스코르브산은 주로 식물과 과일에 많이 함유되어 있다. 식물 종에 따라 아스코르브산의 양은 매우 다양한데, 토마토의 경우 다른 과일에 비해 비교적 낮은 아스코르브산(10-40 mg/100g fw)을 포함하고 있는 것으로 알려져 있다. 따라서 아스코르브산 함량이 증가된 토마토의 개발을 통해 토마토의 영양학적, 산업적 활용도가 크게 개선될 수 있다.Ascorbic acid is a substance acting as vitamin C and is a soluble redox molecule that regulates reactive oxygen specis. Ascorbic acid must be absorbed from the substance. Ascorbic acid is found mainly in plants and fruits. The amount of ascorbic acid varies greatly depending on the plant species, and it is known that tomatoes contain relatively low ascorbic acid (10-40 mg/100g fw) compared to other fruits. Therefore, the nutritional and industrial utilization of tomatoes can be greatly improved through the development of tomatoes with increased ascorbic acid content.
기존 토마토에서 아스코르브산의 함량을 증가시키기 위한 시도는 아스코르브산 합성 유전자의 발현 증가 또는 아스코르브산 분해에 관여하는 효소 활성을 억제를 통해 진행되고 있다. 아스코르브산이 식물세포에서 활성화 산소종을 제거하기 위해서는 주로 Ascorbate Peroxidase (APX)에 의한 촉매 작용이 필요하며, 이 과정에서 아스코르브산은 분해되게 된다. 따라서 토마토에서 APX 유전자의 발현을 낮추게 되면 토마토의 아스코르브산의 함량이 증가될 수 있다. 이때 APX의 발현 감소를 토마토 과실로 제한하게 되면 토마토의 전반적인 발달과정에 있어 아스코르브산 분해를 통한 항산화 기능은 보존되면서 최종 산물인 토마토 과실에서 아스코르브산이 증가하는 결과를 달성할 수 있다. 이를 위해서는 열매에서 특이적으로 기능하는 SlAPX 유전자를 동정하고 그 기능을 검증하는 연구가 필수적이다.Attempts to increase the content of ascorbic acid in the existing tomato are in progress by increasing the expression of an ascorbic acid synthesizing gene or inhibiting the enzyme activity involved in the degradation of ascorbic acid. In order for ascorbic acid to remove reactive oxygen species from plant cells, catalysis mainly by Ascorbate Peroxidase (APX) is required, and in this process, ascorbic acid is decomposed. Therefore, when the expression of the APX gene in tomatoes is lowered, the content of ascorbic acid in tomatoes may be increased. At this time, if the reduction in APX expression is restricted to tomato fruit, the antioxidant function through ascorbic acid decomposition is preserved in the overall development process of tomato, and the result of increasing ascorbic acid in the final product, tomato fruit, can be achieved. To this end, research to identify the SlAPX gene that functions specifically in fruit and to verify its function is essential.
우수한 형질의 식물체를 개발하기 위해 EMS (ethyl methane sulfonate) 또는 감마선 처리 등을 이용하여 자연적 돌연변이를 유도하거나 우수한 양친을 교배하는 전통적 육종방법이 주로 이용되어져 왔으나, 이 방법의 경우 원하는 목표 형질만 바꾸는데 어려움이 있어,최근에는 목표 형질만 대입하기 위해 TALENs, ZFNs, CRISPR/Cas9 등을 이용한 신육종 방법이 적용되고 있다. 그러나 TALENs 또는 ZFNs의 경우 인공적인 뉴클레아제 설계 및 제작에 많은 시간과 비용이 소요되고,다수의 유전자를 편집하기 어려운 단점을 가진다.In order to develop plants with excellent traits, traditional breeding methods such as inducing natural mutations or crossing excellent parents using EMS (ethyl methane sulfonate) or gamma-ray treatment have been mainly used, but in this case, it is difficult to change only the desired target trait. For this reason, recently, a new breeding method using TALENs, ZFNs, CRISPR/Cas9, etc. has been applied to substitute only the target trait. However, in the case of TALENs or ZFNs, it takes a lot of time and money to design and manufacture an artificial nuclease, and it is difficult to edit a large number of genes.
그러나 CRISPR/Cas9 시스템은 매우 높은 표적 특이성을 가지고 있고 메틸화된 DNA도 표적으로 인지할 수 있기 때문에 매우 다양한 유전자에 적용할 수 있으며 멘델의 유전법칙에 따라 자손에게 전달되어 형질의 세대고정이 가능하다는 장점이 있다. 또한, Cas9 단백질과 sgRNA (single guide RNA)만을 필요로 하기 때문에 위의 두 방법에 비해 적은 비용으로 목표 형질의 도입이 가능하다는 장점이 있어 최근 동물 및 식물 등에서 널리 적용되어지고 있는 신육종 기술이다.However, since the CRISPR/Cas9 system has very high target specificity and can recognize methylated DNA as a target, it can be applied to a wide variety of genes. There is this. In addition, since it requires only Cas9 protein and sgRNA (single guide RNA), it has the advantage of being able to introduce target traits at a lower cost compared to the above two methods.
한편, 한국공개특허 제2019-0043841호에는 '식물체에서 CRISPR/Cas9 시스템을 이용하여 LeMADS-RIN 유전자 편집에 의해 에틸렌 생산을 감소시키는 방법'이 개시되어 있고, 한국등록특허 제2090157호에는 '식물체의 초장, 종자 크기 및 출수기를 조절하는 야생벼 유래 APX9 유전자 및 이의 용도'가 개시되어 있으나, 본 발명의 '토마토 열매의 아스코르브산 함량을 조절하는 토마토 유래 APX4 유전자 및 이의 용도'에 대해서는 기재된 바가 없다.On the other hand, Korea Patent Application Publication No. 2019-0043841 discloses 'a method of reducing ethylene production by LeMADS-RIN gene editing in a plant using the CRISPR/Cas9 system', and in Korea Patent No. 2090157, ' Although the wild rice-derived APX9 gene for controlling plant height, seed size and seeding season and its use' has been disclosed, 'a tomato-derived APX4 gene for regulating the content of ascorbic acid in tomato fruit and its use' of the present invention is not described.
본 발명은 상기와 같은 요구에 의해 도출된 것으로서, 본 발명자는 토마토에 존재하는 SlAPX (Solanum lycopersicum Ascorbate Peroxidase) 유전자 중 열매 발달 단계에 특이적으로 발현되는 SlAPX4와 잎에서 높게 발현되는 SlAPX6 유전자를 분리한 후 SlAPX4SlAPX6를 대상으로 각각 세 지역에 작용할 수 있는 VIGS (Virus-Induced Gene Silencing) 벡터를 제작하였고, 제작한 벡터를 immature green 단계의 토마토 열매에 아그로박테리움을 매개로 도입한 후 red-ripeing 단계에서 아스코르브산의 함량을 분석하였다.The present invention was derived from the above needs, and the present inventors isolated SlAPX4 ( Solanum lycopersicum Ascorbate Peroxidase ) genes present in tomato, SlAPX4 specifically expressed in the fruit development stage, and SlAPX6 gene, which is highly expressed in leaves . Afterwards, VIGS (Virus-Induced Gene Silencing) vectors that can act in three regions were prepared for SlAPX4 and SlAPX6 , respectively, and the prepared vector was introduced into immature green tomato fruits through Agrobacterium, followed by red-ripeing. The content of ascorbic acid in the step was analyzed.
그 결과 SlAPX4 유전자 침묵을 유도하는 독립적인 세 가지의 VIGS 벡터를 도입한 토마토 열매의 경우 야생형에 비해 아스코르브산 함량이 유의미하게 증가한 반면, SlAPX6 유전자의 발현을 침묵하는 독립적인 세 가지의 VIGS 벡터를 도입한 토마토 열매의 경우 아스코르브산 함량 증가가 관찰되지 않았다. 이를 통해 열매 특이적 발현을 보이는 SlAPX4 유전자가 아스코르브산 고함량 토마토 개발에 활용될 수 있음을 확인하였다.As a result, in the case of tomato fruit introduced with three independent VIGS vectors inducing SlAPX4 gene silencing, the ascorbic acid content was significantly increased compared to the wild type, whereas three independent VIGS vectors silencing the SlAPX6 gene expression were introduced. No increase in ascorbic acid content was observed in one tomato fruit. Through this, it was confirmed that the SlAPX4 gene showing fruit-specific expression can be utilized for the development of tomatoes with high ascorbic acid content.
그 후, 본 발명자들은 SlAPX4를 표적으로 하는 CRISPR/Cas9 벡터를 구축하여 형질전환 토마토를 개발하였고 형질전환 토마토에서 SlAPX4의 표적 부위 염기서열을 분석하여 토마토 SlAPX4 유전자 서열상에 편집이 일어남을 확인하였으며, 생식이 가능한 시기인 Ripen Red 시기의 야생형 토마토와 유전자 교정 토마토에서 아스코르브산 함량을 비교해 본 결과,야생형 토마토에 비해 SlAPX4 유전자 편집이 일어난 유전자 교정 토마토에서 아스코르브산 함량이 증가하였음을 확인함으로써,본 발명을 완성하였다.After that, the present inventors developed a transgenic tomato by constructing a CRISPR/Cas9 vector targeting SlAPX4 , and by analyzing the nucleotide sequence of the target site of SlAPX4 in the transgenic tomato, it was confirmed that editing occurred on the tomato SlAPX4 gene sequence, As a result of comparing the ascorbic acid content in the wild-type tomato and the gene-modified tomato of the Ripen Red period, which is a reproductive period, the present invention completed.
상기 과제를 해결하기 위해, 본 발명은 서열번호 2의 아미노산 서열로 이루어진 토마토(Solanum lycopersicum) 유래 APX4 (Ascorbate Peroxidase 4) 단백질 코딩 유전자의 발현을 조절하는 단계를 포함하는, 토마토 열매의 아스코르브산 함량 조절 방법을 제공한다.In order to solve the above problems, the present invention comprises the step of regulating the expression of APX4 (Ascorbate Peroxidase 4) protein coding gene derived from tomato ( Solanum lycopersicum ) consisting of the amino acid sequence of SEQ ID NO: 2 Controlling the content of ascorbic acid in tomato fruits provide a way
또한, 본 발명은 (a) 토마토 유래 APX4 단백질 코딩 유전자를 포함하는 재조합 벡터로 토마토 식물세포를 형질전환하는 단계; 및 (b) 상기 형질전환된 토마토 식물세포로부터 형질전환된 토마토 식물체를 재분화하는 단계;를 포함하는 열매의 아스코르브산 함량이 조절된 형질전환 토마토 식물체의 제조방법을 제공한다.In addition, the present invention comprises the steps of (a) transforming tomato plant cells with a recombinant vector containing a tomato-derived APX4 protein coding gene; and (b) re-differentiating the transformed tomato plant from the transformed tomato plant cells; provides a method for producing a transformed tomato plant in which the ascorbic acid content of the fruit is controlled, including.
또한, 본 발명은 상기 방법에 의해 제조된 열매의 아스코르브산 함량이 증가된 형질전환 토마토 식물체 및 이의 형질전환된 종자를 제공한다.In addition, the present invention provides a transgenic tomato plant having an increased content of ascorbic acid in the fruit produced by the above method and a transformed seed thereof.
또한, 본 발명은 서열번호 2의 아미노산 서열로 이루어진 토마토 유래 APX4 단백질을 코딩하는 유전자를 유효성분으로 함유하는 토마토 열매의 아스코르브산 함량 조절용 조성물을 제공한다.In addition, the present invention provides a composition for regulating the content of ascorbic acid in tomato fruits, which contains a tomato-derived APX4 protein encoding gene consisting of the amino acid sequence of SEQ ID NO: 2 as an active ingredient.
또한, 본 발명은 서열번호 30의 염기서열로 이루어진, 토마토 유래 APX4 유전자의 표적 염기서열에 특이적인 가이드 RNA(guide RNA)를 암호화하는 DNA 및 엔도뉴클레아제(endonuclease) 단백질을 암호화하는 핵산 서열을 포함하는 재조합 벡터; 또는 서열번호 30의 염기서열로 이루어진, 토마토 유래 APX4 유전자의 표적 염기서열에 특이적인 가이드 RNA와 엔도뉴클레아제 단백질의 복합체(ribonucleoprotein);를 유효성분으로 함유하는, 토마토 열매의 아스코르브산 함량을 증가시키기 위한 유전체 교정용 조성물을 제공한다.In addition, the present invention, consisting of the nucleotide sequence of SEQ ID NO: 30, DNA encoding a guide RNA (guide RNA) specific to the target nucleotide sequence of the tomato-derived APX4 gene and a nucleic acid sequence encoding an endonuclease protein a recombinant vector comprising; or a complex (ribonucleoprotein) of a guide RNA and an endonuclease protein specific for the target nucleotide sequence of the tomato-derived APX4 gene consisting of the nucleotide sequence of SEQ ID NO: 30; as an active ingredient, increase the ascorbic acid content of tomato fruits It provides a composition for genome editing for
또한, 본 발명은 서열번호 30의 염기서열로 이루어진, 토마토 유래 APX4 유전자의 표적 염기서열에 특이적인 가이드 RNA를 유효성분으로 함유하는, 토마토 열매의 아스코르브산 함량을 증가시키기 위한 유전체 교정용 조성물을 제공한다.In addition, the present invention provides a composition for genome editing for increasing the content of ascorbic acid in tomato fruits, comprising, as an active ingredient, a guide RNA specific for the target nucleotide sequence of the tomato-derived APX4 gene, consisting of the nucleotide sequence of SEQ ID NO: 30 do.
또한, 본 발명은 (a) 서열번호 30의 염기서열로 이루어진, 토마토 유래 APX4 유전자의 표적 염기서열에 특이적인 가이드 RNA 및 엔도뉴클레아제 단백질을 토마토 식물세포에 도입하여 유전체를 교정하는 단계; 및 (b) 상기 유전체가 교정된 토마토 식물세포로부터 토마토 식물체를 재분화하는 단계;를 포함하는, 열매의 아스코르브산 함량이 증가된 유전체 교정 토마토 식물체의 제조방법을 제공한다.In addition, the present invention comprises the steps of (a) introducing a guide RNA and an endonuclease protein specific for the target nucleotide sequence of the tomato-derived APX4 gene consisting of the nucleotide sequence of SEQ ID NO: 30 into tomato plant cells to correct the genome; And (b) the step of redifferentiating the tomato plant from the tomato plant cells in which the genome has been corrected; provides a method for producing a genome-edited tomato plant comprising, the ascorbic acid content of the fruit is increased.
또한, 본 발명은 상기 방법에 의해 제조된 열매의 아스코르브산 함량이 증가된 유전체 교정 토마토 식물체 및 이의 유전체가 교정된 종자를 제공한다.In addition, the present invention provides a genome-corrected tomato plant having an increased content of ascorbic acid in the fruit produced by the above method, and a seed whose genome has been corrected.
본 발명에서 제시한 SlAPX4 유전자 발현 침묵을 통한 토마토 열매 내 아스코르브산 함량 증가 기술은 식물체 내 아스코르브산의 분해를 저해시켜 아스코르브산의 축적을 증가시키므로, 아스코르브산 함량이 증가된 식물체를 개발하는데 이용될 수 있을 것이다. 또한, 본 발명에서 제시한 APX4 유전자 편집 기술은 자연적 변이와 구별할 수 없는 변이를 유도하므로, 안전성과 환경 유해성 여부를 평가하기 위해 막대한 비용과 시간이 소모되는 GMO(Genetically Modified Organism) 작물과 달리 비용과 시간을 절약할 수 있을 것으로 기대된다.The technique of increasing the ascorbic acid content in tomato fruits through SlAPX4 gene expression silencing presented in the present invention increases the accumulation of ascorbic acid by inhibiting the decomposition of ascorbic acid in the plant. There will be. In addition, since the APX4 gene editing technology presented in the present invention induces mutations that cannot be distinguished from natural mutations, it is costly, unlike Genetically Modified Organism (GMO) crops, which consume enormous costs and time to evaluate safety and environmental hazards. and time is expected to be saved.
도 1은 토마토에 존재하는 SlAPX4 (A)와 SlAPX6 (B)의 코딩 서열(coding sequence)을 보여주는 그림이다.1 is a diagram showing the coding sequence of SlAPX4 (A) and SlAPX6 (B) present in tomato.
도 2는 SlAPX4SlAPX6의 토마토 열매 발달 단계별 발현 패턴을 Heinz (Solanum lycopersicum cv. Heinz) 및 마이크로톰 (S. lycopersicum cv. Micro-Tom) 토마토에서 분석한 그래프이다.2 is a graph analyzing the expression patterns of SlAPX4 and SlAPX6 at each stage of tomato fruit development in Heinz ( Solanum lycopersicum cv. Heinz) and S. lycopersicum cv. Micro-Tom tomatoes.
도 3은 SlAPX4SlAPX6의 발현 억제를 위해 제작한 VIGS 벡터의 구조를 도식화한 그림이다.3 is a diagram schematically illustrating the structure of the VIGS vector constructed to suppress the expression of SlAPX4 and SlAPX6 .
도 4는 VIGS 벡터가 도입된 토마토 열매에서 아스코르브산 함량을 측정하여 비교한 그래프이다.4 is a graph comparing ascorbic acid content in tomato fruits introduced with VIGS vector.
도 5는 SlAPX4 유전자에서 표적 서열의 위치(A)와 Cas9 단백질 및 SlAPX4 유전자를 표적하는 sgRNA를 운반하는 재조합 벡터(B)의 구성을 보여준다.5 shows the location of the target sequence in the SlAPX4 gene (A) and the construction of a recombinant vector (B) carrying the Cas9 protein and sgRNA targeting the SlAPX4 gene.
도 6은 개발된 형질전환 식물체 형질의 세대 고정여부를 파악하기 위해 형질전환 식물체 2세대에서 표적 서열 변화 (돌연변이 발생)를 확인한 결과(A)와 SlAPX4 유전자교정 식물체 2세대의 아스코르브산 함량을 측정한 그래프(B)이다.Figure 6 is the result of confirming the target sequence change (mutation) in the second generation of transgenic plants to determine whether the generation of the developed transgenic plant trait is fixed (A) and the ascorbic acid content of the second generation of SlAPX4 gene-corrected plants. It is a graph (B).
본 발명은 서열번호 2의 아미노산 서열로 이루어진 토마토(Solanum lycopersicum) 유래 APX4 (Ascorbate Peroxidase 4) 단백질 코딩 유전자의 발현을 조절하는 단계를 포함하는, 토마토 열매의 아스코르브산 함량 조절 방법을 제공한다.The present invention provides a method for regulating the content of ascorbic acid in tomato fruit, comprising the step of regulating the expression of APX4 (Ascorbate Peroxidase 4) protein-coding gene derived from tomato ( Solanum lycopersicum ) consisting of the amino acid sequence of SEQ ID NO: 2.
본 발명의 일 구현 예에 따른 조절 방법에 있어서, 상기 APX4 단백질은 바람직하게는 서열번호 2의 아미노산 서열로 이루어진 것일 수 있으며, 더욱 바람직하게는 서열번호 2의 아미노산 서열 중 167 내지 250번째 아미노산 서열로 이루어질 수 있으나, 이에 제한되지 않는다. 상기 서열번호 2의 아미노산 서열 중 167 내지 250번째 아미노산 서열은 서열번호 1의 염기서열에서 499 내지 753번째 염기서열에 해당한다.In the control method according to an embodiment of the present invention, the APX4 protein may preferably consist of the amino acid sequence of SEQ ID NO: 2, more preferably, the 167th to 250th amino acid sequence among the amino acid sequence of SEQ ID NO: 2 may be made, but is not limited thereto. The 167th to 250th amino acid sequence of the amino acid sequence of SEQ ID NO: 2 corresponds to the 499th to 753th nucleotide sequence in the nucleotide sequence of SEQ ID NO: 1.
본 발명에 따른 토마토 유래 APX4 단백질의 범위는 서열번호 2로 표시되는 아미노산 서열을 갖는 단백질 및 상기 단백질의 기능적 동등물을 포함한다. 본 발명에 있어서, 용어 "기능적 동등물"이란 아미노산의 부가, 치환 또는 결실의 결과, 상기 서열번호 2로 표시되는 아미노산 서열과 적어도 70% 이상, 바람직하게는 80% 이상, 더욱 바람직하게는 90% 이상, 더 더욱 바람직하게는 95% 이상의 서열 상동성을 갖는 것으로, 서열번호 2로 표시되는 단백질과 실질적으로 동질의 생리활성을 나타내는 단백질을 말한다. "실질적으로 동질의 생리활성"이란 토마토 열매의 아스코르브산 함량을 조절하는 활성을 의미한다.The scope of the tomato-derived APX4 protein according to the present invention includes a protein having the amino acid sequence shown in SEQ ID NO: 2 and functional equivalents of the protein. In the present invention, the term "functional equivalent" means at least 70% or more, preferably 80% or more, more preferably 90% of the amino acid sequence represented by SEQ ID NO: 2 as a result of addition, substitution or deletion of amino acids. As described above, more preferably, it refers to a protein having a sequence homology of 95% or more and exhibiting physiological activity substantially identical to that of the protein represented by SEQ ID NO: 2. "Substantially homogenous physiological activity" means the activity of regulating the content of ascorbic acid in tomato fruit.
본 발명에 따른 토마토 열매의 아스코르브산 함량을 조절하는 방법은, 상기 토마토 유래 APX4 단백질 코딩 유전자의 발현을 식물세포에서 저해시켜 비형질전환 식물체에 비해 열매의 아스코르브산 함량을 증가시키는 것일 수 있으나, 이에 제한되지 않는다.The method for regulating the ascorbic acid content of tomato fruits according to the present invention may be to increase the ascorbic acid content of fruits compared to non-transformed plants by inhibiting the expression of the tomato-derived APX4 protein coding gene in plant cells, but this not limited
본 발명의 일 구현 예에 있어서, 상기 토마토 유래 APX4 단백질을 코딩하는 유전자의 발현 억제는 토마토 유래 APX4 단백질 코딩 유전자에 VIGS (Virus-induced gene silencing) 시스템, RNAi 또는 안티센스 RNA, T-DNA 삽입, 내생 트랜스포존(transposon), X-레이 또는 γ-레이 조사를 통한 돌연변이 유발, 또는 CRISPR/Cas9 유전자 교정 시스템 등을 이용하여 토마토 유래 APX4 단백질 코딩 유전자의 발현을 저해(억제)하는 것일 수 있으나, 이에 제한되지 않으며, 유전자의 발현을 저해하는 당업계의 통상의 방법이면 모두 가능할 수 있다.In one embodiment of the present invention, the inhibition of expression of the gene encoding the tomato-derived APX4 protein is a virus-induced gene silencing (VIGS) system, RNAi or antisense RNA, T-DNA insertion, endogenous to the tomato-derived APX4 protein-coding gene. Transposon, mutagenesis through X-ray or γ-ray irradiation, or inhibiting (suppressing) the expression of the tomato-derived APX4 protein-coding gene using a CRISPR/Cas9 gene correction system, etc., but is not limited thereto And, any conventional method in the art for inhibiting gene expression may be possible.
또한, 본 발명은 상기 토마토 유래 APX4 단백질을 코딩하는 유전자를 제공한다. 상기 토마토 유래 APX4 단백질을 코딩하는 유전자는 게놈 DNA와 cDNA를 모두 포함한다. 바람직하게는, 본 발명의 토마토 유래 APX4 단백질을 코딩하는 유전자는 서열번호 1의 염기서열을 포함할 수 있다. 또한, 상기 염기서열의 상동체가 본 발명의 범위 내에 포함된다. 구체적으로, 상기 유전자는 서열번호 1의 염기서열과 각각 70% 이상, 더욱 바람직하게는 80% 이상, 더 더욱 바람직하게는 90% 이상, 가장 바람직하게는 95% 이상의 서열 상동성을 가지는 염기 서열을 포함할 수 있다. 폴리뉴클레오티드에 대한 "서열 상동성의 %"는 두 개의 최적으로 배열된 서열과 비교 영역을 비교함으로써 확인되며, 비교 영역에서의 폴리뉴클레오티드 서열의 일부는 두 서열의 최적 배열에 대한 참고 서열(추가 또는 삭제를 포함하지 않음)에 비해 추가 또는 삭제(즉, 갭)를 포함할 수 있다.In addition, the present invention provides a gene encoding the tomato-derived APX4 protein. The gene encoding the tomato-derived APX4 protein includes both genomic DNA and cDNA. Preferably, the gene encoding the tomato-derived APX4 protein of the present invention may include the nucleotide sequence of SEQ ID NO: 1. In addition, homologs of the nucleotide sequence are included within the scope of the present invention. Specifically, the gene comprises a nucleotide sequence having at least 70%, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% sequence homology to the nucleotide sequence of SEQ ID NO: 1, respectively. may include The "% sequence homology" for a polynucleotide is determined by comparing two optimally aligned sequences with a comparison region, wherein a portion of the polynucleotide sequence in the comparison region is a reference sequence (additions or deletions) to the optimal alignment of the two sequences. may include additions or deletions (ie, gaps) compared to (not including).
본 명세서에서 용어 "재조합"은 세포가 이종의 핵산을 복제하거나, 상기 핵산을 발현하거나 또는 펩티드, 이종의 펩티드 또는 이종의 핵산에 의해 암호된 단백질을 발현하는 세포를 지칭하는 것이다. 재조합 세포는 상기 세포의 천연 형태에서는 발견되지 않는 유전자 또는 유전자 절편을, 센스 또는 안티센스 형태 중 하나로 발현할 수 있다. 또한 재조합 세포는 천연 상태의 세포에서 발견되는 유전자를 발현할 수 있으며, 그러나 상기 유전자는 변형된 것으로서 인위적인 수단에 의해 세포 내 재도입된 것이다.As used herein, the term “recombinant” refers to a cell in which the cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a peptide, a heterologous peptide or a protein encoded by the heterologous nucleic acid. Recombinant cells can express genes or gene segments not found in the native form of the cell, either in sense or antisense form. Recombinant cells can also express genes found in cells in a natural state, but the genes are modified and re-introduced into cells by artificial means.
용어 "벡터"는 세포 내로 전달하는 DNA 단편(들), 핵산 분자를 지칭할 때 사용된다. 벡터는 DNA를 복제시키고, 숙주세포에서 독립적으로 재생산될 수 있다. 용어 "전달체"는 흔히 "벡터"와 호환하여 사용된다. 용어 "발현 벡터"는 목적한 코딩 서열과, 특정 숙주 생물에서 작동가능하게 연결된 코딩 서열을 발현하는데 필수적인 적정 핵산 서열을 포함하는 재조합 DNA 분자를 의미한다.The term “vector” is used to refer to a DNA fragment(s), a nucleic acid molecule, that is delivered into a cell. The vector replicates DNA and can be reproduced independently in a host cell. The term "carrier" is often used interchangeably with "vector." The term "expression vector" refers to a recombinant DNA molecule comprising a desired coding sequence and a suitable nucleic acid sequence necessary for expression of an operably linked coding sequence in a particular host organism.
본 발명의 상기 벡터는 전형적으로 클로닝 또는 발현을 위한 벡터로서 구축될 수 있다. 또한, 본 발명의 벡터는 원핵 세포 또는 진핵 세포를 숙주로 하여 구축될 수 있다. 예를 들어, 본 발명의 벡터가 발현 벡터이고, 원핵 세포를 숙주로 하는 경우에는, 전사를 진행시킬 수 있는 강력한 프로모터 (예컨대, pLλ프로모터, Trp 프로모터, Lac 프로모터, T7 프로모터, Tac 프로모터 등), 해독의 개시를 위한 리보좀 결합 자리 및 전사/해독 종결 서열을 포함하는 것이 일반적이다. 숙주 세포로서 대장균(Escherichia coli)이 이용되는 경우, E. coli 트립토판 생합성 경로의 프로모터 및 오퍼레이터 부위, 그리고 파아지 λ의 좌향 프로모터 (pLλ프로모터)가 조절 부위로서 이용될 수 있다.The vector of the present invention can typically be constructed as a vector for cloning or expression. In addition, the vector of the present invention can be constructed using a prokaryotic cell or a eukaryotic cell as a host. For example, when the vector of the present invention is an expression vector and a prokaryotic cell is used as a host, a strong promoter capable of propagating transcription (eg, pLλ promoter, Trp promoter, Lac promoter, T7 promoter, Tac promoter, etc.); It generally includes a ribosome binding site for initiation of translation and a transcription/translation termination sequence. When Escherichia coli is used as the host cell, the promoter and operator sites of the E. coli tryptophan biosynthesis pathway, and the left-handed promoter (pLλ promoter) of phage λ may be used as regulatory regions.
본 발명의 재조합 벡터에서, 상기 프로모터는 형질전환에 적합한 프로모터들로서, 바람직하게는 CaMV 35S 프로모터, 액틴 프로모터, 유비퀴틴 프로모터, pEMU 프로모터, MAS 프로모터 또는 히스톤 프로모터일 수 있으며, 바람직하게는 CaMV 35S 프로모터일 수 있으나, 이에 제한되지 않는다.In the recombinant vector of the present invention, the promoter is a promoter suitable for transformation, preferably CaMV 35S promoter, actin promoter, ubiquitin promoter, pEMU promoter, MAS promoter or histone promoter, preferably CaMV 35S promoter. However, it is not limited thereto.
본 발명에 있어서, "프로모터"란 용어는 구조 유전자로부터의 DNA 업스트림의 영역을 의미하며 전사를 개시하기 위하여 RNA 폴리머라아제가 결합하는 DNA 분자를 말한다. "식물 프로모터"는 식물 세포에서 전사를 개시할 수 있는 프로모터이다. "항시성(constitutive) 프로모터"는 대부분의 환경 조건 및 발달 상태 또는 세포 분화하에서 활성이 있는 프로모터이다. 형질전환체의 선택이 각종 단계에서 각종 조직에 의해서 이루어질 수 있기 때문에 항시성 프로모터가 본 발명에서 바람직할 수 있다. 따라서, 항시성 프로모터는 선택 가능성을 제한하지 않는다.In the present invention, the term "promoter" refers to a region upstream of DNA from a structural gene and refers to a DNA molecule to which RNA polymerase binds to initiate transcription. A “plant promoter” is a promoter capable of initiating transcription in a plant cell. A "constitutive promoter" is a promoter that is active under most environmental conditions and developmental states or cell differentiation. A constitutive promoter may be preferred in the present invention since the selection of transformants can be made by various tissues at various stages. Thus, constitutive promoters do not limit the selectivity.
본 발명의 재조합 벡터는 당업자에 주지된 방법에 의해 구축될 수 있다. 상기 방법은 시험관 내 재조합 DNA 기술, DNA 합성 기술 및 생체 내 재조합 기술 등을 포함한다. 상기 DNA 서열은 mRNA 합성을 이끌기 위해 발현 벡터 내의 적당한 프로모터에 효과적으로 연결될 수 있다. 또한 벡터는 번역 개시 부위로서 리보솜 결합 부위 및 전사 터미네이터를 포함할 수 있다.The recombinant vector of the present invention can be constructed by methods well known to those skilled in the art. The method includes in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology. The DNA sequence can be effectively linked to a suitable promoter in an expression vector to direct mRNA synthesis. The vector may also include a ribosome binding site as a translation initiation site and a transcription terminator.
본 발명에 따른 토마토 열매의 아스코르브산 함량 조절 방법에 있어서, 재조합 벡터의 바람직한 예는 VIGS 벡터 또는 RNAi 벡터이다. VIGS는 바이러스 벡터에 식물 유전자를 도입한 후 식물체를 감염시키면, 그 도입된 유전자의 내인성 유전자가 발현이 억제되는 현상을 말한다. 이는 PTGS (Post-transcriptional gene silencing)의 일종으로서, 전사-후 (post-transcriptional), RNA 턴오버 (RNA turnover) 및 뉴클레오티드 서열 특이적 (nucleotide sequence-specific) 이라는 특징들을 가진다. 상기 VIGS 벡터는 외래 유전자를 도입한 식물체 내에서 일시적으로 발현시킬 수 있는 일시적 (transient) 발현 벡터 및 외래 유전자가 도입된 식물체에서 영구적으로 발현시킬 수 있는 식물 발현 벡터로 사용할 수 있다.In the method for controlling the content of ascorbic acid in tomato fruit according to the present invention, a preferred example of the recombinant vector is a VIGS vector or an RNAi vector. VIGS refers to a phenomenon in which expression of an endogenous gene of the introduced gene is suppressed when a plant is infected after introducing a plant gene into a viral vector. It is a type of post-transcriptional gene silencing (PTGS), and has characteristics of post-transcriptional, RNA turnover, and nucleotide sequence-specific. The VIGS vector can be used as a transient expression vector that can be temporarily expressed in a plant into which a foreign gene is introduced, and a plant expression vector that can be permanently expressed in a plant into which the foreign gene is introduced.
식물 발현 벡터의 바람직한 예는 아그로박테리움 투머파시엔스 (Agrobacterium tumefaciens)와 같은 적당한 숙주에 존재할 때 그 자체의 일부, 소위 T-영역을 식물 세포로 전이시킬 수 있는 Ti-플라스미드 벡터이다. 다른 유형의 Ti-플라스미드 벡터 (EP 0 116 718 B1호 참조)는 현재 식물 세포, 또는 잡종 DNA를 식물의 게놈 내에 적당하게 삽입시키는 새로운 식물이 생산될 수 있는 원형질체로 잡종 DNA 서열을 전이시키는데 이용되고 있다. Ti-플라스미드 벡터의 특히 바람직한 형태는 EP 0 120 516 B1호 및 미국 특허 제4,940,838호에 청구된 바와 같은 소위 바이너리 (binary) 벡터이다. 본 발명에 따른 DNA를 식물 숙주에 도입시키는데 이용될 수 있는 다른 적합한 벡터는 이중 가닥 식물 바이러스 (예를 들면, CaMV) 및 단일 가닥 바이러스, 게미니 바이러스 등으로부터 유래될 수 있는 것과 같은 바이러스 벡터, 예를 들면 비완전성 식물 바이러스 벡터로부터 선택될 수 있다. 그러한 벡터의 사용은 특히 식물 숙주를 적당하게 형질전환하는 것이 어려울 때 유리할 수 있다.A preferred example of a plant expression vector is a Ti-plasmid vector capable of transferring a part of itself, the so-called T-region, into a plant cell when present in a suitable host such as Agrobacterium tumefaciens . Another type of Ti-plasmid vector (see EP 0 116 718 B1) is currently used to transfer hybrid DNA sequences into plant cells, or protoplasts from which new plants can be produced that properly insert the hybrid DNA into the genome of the plant and have. A particularly preferred form of the Ti-plasmid vector is the so-called binary vector as claimed in EP 0 120 516 B1 and US Pat. No. 4,940,838. Other suitable vectors that can be used to introduce the DNA according to the invention into a plant host are viral vectors such as those that can be derived from double-stranded plant viruses (eg CaMV) and single-stranded viruses, gemini viruses, etc. For example, it may be selected from an incomplete plant viral vector. The use of such vectors can be advantageous, especially when it is difficult to adequately transform a plant host.
재조합 발현 벡터는 바람직하게는 하나 이상의 선택성 마커를 포함할 수 있다. 상기 마커는 통상적으로 화학적인 방법으로 선택될 수 있는 특성을 갖는 핵산 서열로, 형질전환된 세포를 비형질전환 세포로부터 구별할 수 있는 모든 유전자가 이에 해당된다. 상기 마커 유전자는 항생제 저항성 유전자(dominant drug resistance gene)일 수 있으나, 이에 제한되지 않는다.The recombinant expression vector may preferably comprise one or more selectable markers. The marker is a nucleic acid sequence having a characteristic that can be selected by a conventional chemical method, and includes all genes capable of distinguishing a transformed cell from a non-transformed cell. The marker gene may be an antibiotic resistance gene (dominant drug resistance gene), but is not limited thereto.
본 발명의 벡터를 안정되면서 연속적으로 클로닝 및 발현시킬 수 있는 숙주세포는 미세조류, 미생물 등을 포함한 당업계에 공지된 어떠한 숙주세포도 이용할 수 있으며, 예컨대, E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, 바실러스 서브틸리스(Bacillus subtilis), 바실러스 츄린겐시스(B. thuringiensis)와 같은 바실러스 속 균주, 그리고 살모넬라 티피무리움(Salmonella typhimurium), 세라티아 마르세슨스(Serratia marcescens) 및 다양한 슈도모나스(Pseudomonas) 종과 같은 장내균과 균주 등이 있다.As a host cell capable of stably and continuously cloning and expressing the vector of the present invention, any host cell known in the art, including microalgae and microorganisms, may be used, for example, E. coli JM109, E. coli BL21, Bacillus genus strains such as E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis , B. thuringiensis , And Salmonella typhimurium ( Salmonella typhimurium ), Serratia marcescens ( Serratia marcescens ) and various Pseudomonas enterobacteriaceae and strains, such as (Pseudomonas) species.
또한, 본 발명의 벡터를 진핵 세포에 형질전환시키는 경우에는 숙주세포로서, 효모 (예컨대, Saccharomyce cerevisiae), 곤충세포, 사람세포 (예컨대, CHO 세포주(Chinese hamster ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN 및 MDCK 세포주) 및 식물세포 등이 이용될 수 있으며, 바람직하게는 식물세포이다.In addition, when the vector of the present invention is transformed into eukaryotic cells, as host cells, yeast (eg, Saccharomyce cerevisiae ), insect cells, human cells (eg, CHO cell line (Chinese hamster ovary), W138, BHK, COS-7) , 293, HepG2, 3T3, RIN and MDCK cell lines) and plant cells may be used, preferably plant cells.
식물의 형질전환은 DNA를 식물에 전이시키는 임의의 방법을 의미한다. 그러한 형질전환 방법은 반드시 재생 및 (또는) 조직 배양기간을 가질 필요는 없다. 식물 종의 형질전환은 이제는 쌍자엽 식물뿐만 아니라 단자엽 식물 양자를 포함한 식물 종에 대해 일반적이다. 원칙적으로, 임의의 형질전환 방법은 본 발명에 따른 잡종 DNA를 적당한 선조 세포로 도입시키는데 이용될 수 있다. 방법은 원형질체에 대한 칼슘/폴리에틸렌 글리콜 방법(Krens, F.A. et al., 1982, Nature 296, 72-74; Negrutiu I. et al., 1987, Plant Mol. Biol. 8, 363-373), 원형질체의 전기천공법(Shillito R.D. et al., 1985 Bio/Technol. 3, 1099-1102), 식물 요소로의 현미주사법(Crossway A. et al., 1986, Mol. Gen. Genet. 202, 179-185), 각종 식물 요소의 (DNA 또는 RNA-코팅된) 입자충격법(Klein T.M. et al., 1987, Nature 327, 70), 식물의 침윤 또는 성숙 화분 또는 소포자의 형질전환에 의한 아그로박테리움 튜머파시엔스 매개된 유전자 전이에서 (비완전성) 바이러스에 의한 감염(EP 0 301 316호) 등으로부터 적당하게 선택될 수 있다. 본 발명에 따른 바람직한 방법은 아그로박테리움 매개된 DNA 전달을 포함한다.Transformation of a plant refers to any method of transferring DNA into a plant. Such transformation methods need not necessarily have a period of regeneration and/or tissue culture. Transformation of plant species is now common for plant species including both monocots as well as dicots. In principle, any transformation method can be used to introduce the hybrid DNA according to the invention into suitable progenitor cells. Methods include the calcium/polyethylene glycol method for protoplasts (Krens, F.A. et al., 1982, Nature 296, 72-74; Negrutiu I. et al., 1987, Plant Mol. Biol. 8, 363-373), protoplasts. Electroporation (Shillito R.D. et al., 1985 Bio/Technol. 3, 1099-1102), microinjection with plant elements (Crossway A. et al., 1986, Mol. Gen. Genet. 202, 179-185) , Agrobacterium tumefaciens by particle bombardment of various plant elements (DNA or RNA-coated) (Klein T.M. et al., 1987, Nature 327, 70), infiltration of plants or transformation of mature pollen or vesicles. In mediated gene transfer, it can be appropriately selected from (incomplete) viral infection (EP 0 301 316) and the like. A preferred method according to the present invention comprises Agrobacterium mediated DNA delivery.
본 발명은 또한,The present invention also
(a) 토마토 유래 APX4 단백질 코딩 유전자를 포함하는 재조합 벡터로 토마토 식물세포를 형질전환하는 단계; 및(a) transforming tomato plant cells with a recombinant vector containing a tomato-derived APX4 protein-coding gene; and
(b) 상기 형질전환된 토마토 식물세포로부터 형질전환된 토마토 식물체를 재분화하는 단계;를 포함하는 열매의 아스코르브산 함량이 조절된 형질전환 토마토 식물체의 제조방법을 제공한다.(b) redifferentiating the transformed tomato plant from the transformed tomato plant cells; provides a method for producing a transformed tomato plant in which the ascorbic acid content of the fruit is regulated, including.
본 발명의 형질전환 토마토 식물체의 제조방법에 있어서, 상기 토마토 유래 APX4 단백질은 바람직하게는 서열번호 2의 아미노산 서열로 이루어진 단백질 및 상기 단백질의 기능적 동등물을 포함할 수 있고, 구체적인 내용은 전술한 것과 같다.In the method for producing a transgenic tomato plant of the present invention, the tomato-derived APX4 protein may include a protein consisting of the amino acid sequence of SEQ ID NO: 2 and a functional equivalent of the protein, and the specific details are as described above same.
또한, 본 발명의 형질전환 토마토 식물체의 제조방법에 있어서, 상기 식물세포를 형질전환시키는 방법은 전술한 바와 같으며, 상기 형질전환된 식물세포로부터 형질전환 식물을 재분화하는 방법은 당업계에 공지된 임의의 방법을 이용할 수 있다. 형질전환된 식물세포는 전식물로 재분화되어야 한다. 캘러스 또는 원형질체 배양으로부터 성숙한 식물의 재분화를 위한 기술은 수많은 여러 가지 종에 대해서 당업계에 주지되어 있다.In addition, in the method for producing a transgenic tomato plant of the present invention, the method for transforming the plant cell is as described above, and the method for redifferentiating the transgenic plant from the transformed plant cell is known in the art. Any method may be used. Transformed plant cells must be redifferentiated into whole plants. Techniques for the redifferentiation of mature plants from callus or protoplast cultures are well known in the art for a number of different species.
식물의 형질전환에 이용되는 "식물세포"는 어떤 식물세포도 된다. 식물세포는 배양 세포, 배양 조직, 배양기관 또는 전체 식물이다. "식물 조직"은 분화된 또는 미분화된 식물의 조직, 예를 들면 이에 한정되진 않으나, 뿌리, 줄기, 잎, 꽃가루, 종자, 암 조직 및 배양에 이용되는 다양한 형태의 세포들, 즉 단일 세포, 원형질체(protoplast), 싹 및 캘러스 조직을 포함한다. 식물 조직은 인 플란타(in planta)이거나 기관 배양, 조직배양 또는 세포 배양 상태일 수 있다.The "plant cell" used for transformation of a plant may be any plant cell. Plant cells are cultured cells, cultured tissues, cultured organs or whole plants. "Plant tissue" refers to differentiated or undifferentiated plant tissues, such as, but not limited to, roots, stems, leaves, pollen, seeds, cancer tissues and various types of cells used in culture, ie, single cells, protoplasts. (protoplast), shoots and callus tissue. The plant tissue may be in planta or in an organ culture, tissue culture or cell culture state.
본 발명의 일 구현 예에 따른 형질전환 토마토 식물체의 제조방법에 있어서, 상기 토마토 유래 APX4 단백질을 코딩하는 유전자의 발현을 토마토 식물세포에서 저해시키면 비형질전환 식물체 즉, 야생형 토마토에 비해 토마토 식물체의 열매 내 아스코르브산 함량이 증가될 수 있으나, 이에 제한되지 않는다.In the method for producing a transgenic tomato plant according to an embodiment of the present invention, when the expression of the tomato-derived APX4 protein-encoding gene is inhibited in tomato plant cells, the non-transformed plant, that is, the fruit of the tomato plant compared to the wild-type tomato My ascorbic acid content may be increased, but is not limited thereto.
상기 토마토 유래 APX4 단백질 코딩 유전자의 발현 저해는 토마토 유래 APX4 유전자에 대한 센스(sense) 또는 안티센스(antisense) DNA, 또는 microRNA를 포함하는 재조합 벡터로 토마토 식물세포를 형질전환시켜 APX4 단백질 코딩 유전자의 발현을 저해하는 것일 수 있으나, 이에 제한되지 않으며, 당업계에 공지된 유전자 발현 저해 기술을 이용할 수 있다.The tomato-derived APX4 protein coding Inhibition of gene expression may be to inhibit the expression of APX4 protein-coding gene by transforming tomato plant cells with a recombinant vector containing sense or antisense DNA, or microRNA for the tomato-derived APX4 gene. It is not limited thereto, and a gene expression inhibition technique known in the art may be used.
본 발명은 또한, 상기 형질전환 토마토 식물체의 제조방법에 의해 제조된 열매의 아스코르브산 함량이 증가된 형질전환 토마토 식물체 및 이의 형질전환된 종자를 제공한다.The present invention also provides a transgenic tomato plant having an increased content of ascorbic acid in the fruit produced by the method for producing the transgenic tomato plant, and a transformed seed thereof.
본 발명에 따른 열매의 아스코르브산 함량이 증가된 형질전환 토마토 식물체는 APX4 단백질 코딩 유전자의 발현을 저해시켜 열매 내 아스코르브산의 함량이 증가된 것을 특징으로 한다.The transgenic tomato plant with an increased content of ascorbic acid in the fruit according to the present invention is characterized in that the content of ascorbic acid in the fruit is increased by inhibiting the expression of the APX4 protein coding gene.
본 발명은 또한, 서열번호 2의 아미노산 서열로 이루어진 토마토 유래 APX4 단백질을 코딩하는 유전자를 유효성분으로 함유하는 토마토 열매의 아스코르브산 함량 조절용 조성물을 제공한다. 본 발명의 토마토 열매의 아스코르브산 함량 조절용 조성물은 유효성분으로 토마토 열매의 아스코르브산 함량을 조절할 수 있는 토마토 유래 APX4 단백질 코딩 유전자를 포함하며, 상기 유전자의 발현이 저해되면, 토마토 열매의 아스코르브산 함량을 증가시킬 수 있다.The present invention also provides a composition for regulating the content of ascorbic acid in tomato fruits, which contains a tomato-derived APX4 protein encoding gene consisting of the amino acid sequence of SEQ ID NO: 2 as an active ingredient. The composition for controlling the ascorbic acid content of tomato fruits of the present invention includes a tomato-derived APX4 protein coding gene capable of controlling the ascorbic acid content of tomato fruits as an active ingredient, and when the expression of the gene is inhibited, the ascorbic acid content of tomato fruits is reduced can increase
본 발명은 또한, 서열번호 30의 염기서열로 이루어진, 토마토 유래 APX4 유전자의 표적 염기서열에 특이적인 가이드 RNA(guide RNA)를 암호화하는 DNA 및 엔도뉴클레아제(endonuclease) 단백질을 암호화하는 핵산 서열을 포함하는 재조합 벡터; 또는 서열번호 30의 염기서열로 이루어진, 토마토 유래 APX4 유전자의 표적 염기서열에 특이적인 가이드 RNA와 엔도뉴클레아제 단백질의 복합체(ribonucleoprotein);를 유효성분으로 함유하는, 토마토 열매의 아스코르브산 함량을 증가시키기 위한 유전체 교정용 조성물을 제공한다.The present invention also provides a nucleic acid sequence encoding a guide RNA (guide RNA) specific to the target nucleotide sequence of the tomato-derived APX4 gene, and a nucleic acid sequence encoding an endonuclease protein, consisting of the nucleotide sequence of SEQ ID NO: 30 a recombinant vector comprising; or a complex (ribonucleoprotein) of a guide RNA and an endonuclease protein specific for the target nucleotide sequence of the tomato-derived APX4 gene consisting of the nucleotide sequence of SEQ ID NO: 30; as an active ingredient, increase the ascorbic acid content of tomato fruits It provides a composition for genome editing for
본 명세서에서 용어 "유전체/유전자 교정(genome/gene editing)"은, 인간 세포를 비롯한 동·식물 세포의 유전체 염기서열에 표적지향형 변이를 도입할 수 있는 기술로서, DNA 절단에 의한 하나 이상의 핵산 분자의 결실(deletion), 삽입(insertion), 치환(substitutions) 등에 의하여 특정 유전자를 녹-아웃(knock-out) 또는 녹-인(knock-in)하거나, 단백질을 생성하지 않는 비-코딩(non-coding) DNA 서열에도 변이를 도입할 수 있는 기술을 말한다. 본 발명의 목적상 상기 유전체 교정은 특히 엔도뉴클레아제(endonuclease) 예컨대, Cas9 (CRISPR associated protein 9) 단백질 및 가이드 RNA를 이용하여 식물체에 변이를 도입하는 것일 수 있다. 또한, '유전자 교정'은 '유전자 편집'과 혼용되어 사용될 수 있다.As used herein, the term “genome/gene editing” refers to a technology capable of introducing a target-directed mutation into the genome sequence of animal and plant cells, including human cells, and one or more nucleic acid molecules by DNA cleavage. Knock-out or knock-in a specific gene by deletion, insertion, substitution, etc. of coding) refers to a technology that can introduce mutations into DNA sequences. For the purpose of the present invention, the genome editing may be, in particular, introducing a mutation into a plant using an endonuclease, such as a Cas9 (CRISPR associated protein 9) protein and a guide RNA. Also, 'gene editing' may be used interchangeably with 'gene editing'.
또한, 용어 "표적 유전자"는 본 발명을 통해 교정하고자 하는 식물체의 유전체 내에 있는 일부 DNA를 의미하며, 그 유전자의 종류에 제한되지 않으며, 코딩 영역 및 비-코딩 영역을 모두 포함할 수 있다. 당업자는 그 목적에 따라, 제조하고자 하는 유전체 교정 식물체에 대하여 원하는 변이에 따라 상기 표적 유전자를 선별할 수 있다.In addition, the term "target gene" refers to some DNA in the genome of a plant to be corrected through the present invention, is not limited to the type of the gene, and may include both a coding region and a non-coding region. A person skilled in the art can select the target gene according to the desired mutation for the genome-corrected plant to be prepared, depending on the purpose.
또한, 용어 "가이드 RNA(guide RNA)"는 짧은 단일 가닥의 RNA로, 표적 유전자를 암호화하는 염기서열 중 표적 DNA에 특이적인 RNA를 의미하며, 표적 DNA 염기서열과 전부 또는 일부가 상보적으로 결합하여 해당 표적 DNA 염기서열로 엔도뉴클레아제 단백질을 이끄는 역할을 하는 리보핵산을 의미한다. 상기 가이드 RNA는 두 개의 RNA, 즉, crRNA (CRISPR RNA) 및 tracrRNA (trans-activating crRNA)를 구성 요소로 포함하는 이중 RNA (dual RNA); 또는 표적 유전자 내 염기서열과 전부 또는 일부 상보적인 서열을 포함하는 제1 부위 및 엔도뉴클레아제(특히, RNA-가이드 뉴클레아제)와 상호작용하는 서열을 포함하는 제2 부위를 포함하는 단일 사슬 가이드 RNA(single guide RNA, sgRNA) 형태를 말하나, 엔도뉴클레아제가 표적 염기서열에서 활성을 가질 수 있는 형태라면 제한없이 본 발명의 범위에 포함될 수 있으며, 함께 사용된 엔도뉴클레아제의 종류 또는 엔도뉴클레아제의 유래 미생물 등을 고려하여 당업계의 공지된 기술에 따라 제조하여 사용할 수 있다.In addition, the term “guide RNA” is a short single-stranded RNA, and refers to an RNA specific for a target DNA among nucleotide sequences encoding a target gene, and all or part of the target DNA nucleotide sequence and all or part of the nucleotide sequence are complementary Thus, it refers to a ribonucleic acid that leads the endonuclease protein to the corresponding target DNA sequence. The guide RNA may be a dual RNA comprising two RNAs, ie, crRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA); or a single chain comprising a first site comprising a sequence that is all or partly complementary to a nucleotide sequence in a target gene and a second site comprising a sequence that interacts with an endonuclease (especially an RNA-guided nuclease) Refers to the form of guide RNA (single guide RNA, sgRNA), but as long as the endonuclease has activity in the target nucleotide sequence, it may be included in the scope of the present invention without limitation, and the type or endonuclease used together It can be prepared and used according to a technique known in the art in consideration of the microorganisms derived from the nuclease and the like.
또한, 상기 가이드 RNA는 플라스미드 주형으로부터 전사된 것, 생체 외(in vitro)에서 전사된(transcribed) 것(예컨대, 올리고뉴클레오티드 이중가닥) 또는 합성한 가이드 RNA 등일 수 있으나, 이에 제한되지 않는다.In addition, the guide RNA may be transcribed from a plasmid template, transcribed in vitro (eg, oligonucleotide double-stranded), or synthesized guide RNA, but is not limited thereto.
또한, 본 발명에 따른 유전체 교정용 조성물에 있어서, 상기 엔도뉴클레아제 단백질은 Cas9, Cpf1 (also known as Cas12a), TALEN (Transcription activator-like effector nuclease), ZFN (Zinc Finger Nuclease) 또는 이의 기능적 유사체로 이루어진 군으로부터 선택되는 하나 이상일 수 있고, 바람직하게는 RNA-가이드 뉴클레아제인 Cas9 또는 Cpf1 등일 수 있으며, 더욱 바람직하게는 Cas9 단백질일 수 있으나, 이에 제한되지 않는다.In addition, in the composition for genome editing according to the present invention, the endonuclease protein is Cas9, Cpf1 (also known as Cas12a), TALEN (Transcription activator-like effector nuclease), ZFN (Zinc Finger Nuclease) or a functional analog thereof. It may be at least one selected from the group consisting of, preferably, an RNA-guided nuclease, such as Cas9 or Cpf1, and more preferably, a Cas9 protein, but is not limited thereto.
또한, 상기 Cas9 단백질은 스트렙토코커스 피요제네스(Streptococcus pyogenes) 유래의 Cas9 단백질, 캠필로박터 제주니(Campylobacter jejuni) 유래의 Cas9 단백질, 스트렙토코커스 써모필러스(S. thermophilus) 또는 스트렙토코커스 아우레우스(S. aureus) 유래의 Cas9 단백질, 네이쎄리아 메닝기티디스(Neisseria meningitidis) 유래의 Cas9 단백질, 파스투렐라 물토시다(Pasteurella multocida) 유래의 Cas9 단백질, 프란시셀라 노비시다(Francisella novicida) 유래의 Cas9 단백질 등으로 이루어진 군에서 선택된 하나 이상일 수 있으나, 이에 제한되지 않는다. Cas9 단백질 또는 이의 유전자 정보는 NCBI(National Center for Biotechnology Information)의 GenBank와 같은 공지의 데이터베이스에서 얻을 수 있다. 상기 Cas9 유전자 정보는 공지된 서열을 그대로 사용할 수도 있고, 형질도입되는 대상(유기체)의 코돈에 최적화된 서열을 사용할 수 있으나, 이에 제한되지 않는다.In addition, the Cas9 protein is a Cas9 protein derived from Streptococcus pyogenes , a Cas9 protein derived from Campylobacter jejuni , S. thermophilus or Streptococcus aureus ( S. aureus )-derived Cas9 protein, Neisseria meningitidis -derived Cas9 protein, Pasteurella multocida -derived Cas9 protein, Francisella novicida ) derived Cas9 protein, etc. It may be one or more selected from the group consisting of, but is not limited thereto. Cas9 protein or genetic information thereof can be obtained from a known database such as GenBank of the National Center for Biotechnology Information (NCBI). As the Cas9 gene information, a known sequence may be used as it is, or a sequence optimized for the codon of the subject (organism) to be transduced may be used, but is not limited thereto.
Cas9 단백질은 RNA-guided DNA 엔도뉴클레아제 효소로, 이중 가닥 DNA 절단(double stranded DNA break)을 유도한다. Cas9 단백질이 정확하게 표적 염기서열에 결합하여 DNA 가닥을 잘라내기 위해서는 PAM (Protospacer Adjacent Motif)이라 알려진 3개의 염기로 이루어진 짧은 염기서열이 표적 염기서열 옆에 존재해야 하며, Cas9 단백질은 PAM 서열(NGG)로부터 3번째와 4번째 염기쌍 사이를 추정하여 절단한다.Cas9 protein is an RNA-guided DNA endonuclease enzyme that induces double-stranded DNA breaks. In order for the Cas9 protein to accurately bind to the target sequence and cut the DNA strand, a short sequence of three bases known as PAM (Protospacer Adjacent Motif) must exist next to the target sequence, and the Cas9 protein has a PAM sequence (NGG). cleavage by inferring between the 3rd and 4th base pairs from
본 발명에 따른 유전체 교정용 조성물에 있어서, 상기 가이드 RNA와 엔도뉴클레아제 단백질은 리보핵산-단백질(ribonucleoprotein) 복합체를 형성하여 RNA 유전자 가위(RNA-Guided Engineered Nuclease, RGEN)로 작동할 수 있다.In the composition for genome editing according to the present invention, the guide RNA and the endonuclease protein form a ribonucleoprotein complex to operate as RNA-Guided Engineered Nuclease (RGEN).
본 발명의 일 구현 예에 따른 유전체 교정용 조성물에 있어서, 상기 토마토 유래 APX4 유전자는 서열번호 1 또는 서열번호 38의 염기서열로 이루어진 것일 수 있으나, 이에 제한되지 않는다.In the composition for genome editing according to an embodiment of the present invention, the tomato-derived APX4 gene may consist of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 38, but is not limited thereto.
본 발명에 따른 유전체 교정용 조성물에 있어서, 사용된 CRISPR/Cas9 시스템은 교정하고자 하는 특정 유전자의 특정위치에 이중나선 절단을 도입하여 DNA 수선 과정에서 유도되는 불완전 수선에 의한 삽입-결실(insertion-deletion, InDel) 돌연변이를 유도시키는 NHEJ(non-homologous end joining) 기작에 의한 유전자 교정 방법이다.In the composition for genome editing according to the present invention, the CRISPR/Cas9 system used introduces a double helix break at a specific position of a specific gene to be corrected, and an insertion-deletion caused by incomplete repair induced in the DNA repair process. , InDel) is a gene editing method by NHEJ (non-homologous end joining) mechanism that induces mutations.
또한, 본 발명은 서열번호 30의 염기서열로 이루어진, 토마토 유래 APX4 유전자의 표적 염기서열에 특이적인 가이드 RNA를 유효성분으로 함유하는, 토마토 열매의 아스코르브산 함량을 증가시키기 위한 유전체 교정용 조성물을 제공한다.In addition, the present invention provides a genome editing composition for increasing the content of ascorbic acid in tomato fruits, comprising, as an active ingredient, a guide RNA specific for the target nucleotide sequence of the tomato-derived APX4 gene, consisting of the nucleotide sequence of SEQ ID NO: 30 do.
본 발명의 가이드 RNA는 바람직하게는 단일 사슬 가이드 RNA(single guide RNA, sgRNA) 형태일 수 있으나, 이에 제한되지 않으며, sgRNA는 표적 염기서열에 특이적인 가이드 RNA 외에 엔도뉴클레아제 결합 서열에 특이적인 RNA를 포함할 수 있으며, 바람직하게는 상기 가이드 RNA는 서열번호 37의 염기서열로 이루어질 수 있으나, 이에 제한되지 않는다.The guide RNA of the present invention may preferably be in the form of a single guide RNA (sgRNA), but is not limited thereto, and the sgRNA is specific for an endonuclease binding sequence in addition to a guide RNA specific for a target sequence It may include RNA, and preferably, the guide RNA may consist of the nucleotide sequence of SEQ ID NO: 37, but is not limited thereto.
본 발명은 또한,The present invention also
(a) 서열번호 30의 염기서열로 이루어진, 토마토 유래 APX4 유전자의 표적 염기서열에 특이적인 가이드 RNA 및 엔도뉴클레아제 단백질을 토마토 식물세포에 도입하여 유전체를 교정하는 단계; 및(a) correcting the genome by introducing a guide RNA and an endonuclease protein specific for the target nucleotide sequence of the tomato-derived APX4 gene, consisting of the nucleotide sequence of SEQ ID NO: 30, into tomato plant cells; and
(b) 상기 유전체가 교정된 토마토 식물세포로부터 토마토 식물체를 재분화하는 단계;를 포함하는, 열매의 아스코르브산 함량이 증가된 유전체 교정 토마토 식물체의 제조방법을 제공한다.(b) re-differentiating the tomato plant from the tomato plant cells in which the genome has been corrected; provides a method for producing a genome-edited tomato plant comprising, including, the ascorbic acid content of the fruit increased.
본 발명의 유전체 교정 토마토 식물체의 제조방법에 있어서, 상기 (a) 단계의 가이드 RNA 및 엔도뉴클레아제 단백질을 토마토 식물세포에 도입하는 것은, 서열번호 30의 염기서열로 이루어진, 토마토 유래 APX4 유전자의 표적 염기서열에 특이적인 가이드 RNA를 암호화하는 DNA 및 엔도뉴클레아제 단백질을 암호화하는 핵산 서열을 포함하는 재조합 벡터; 또는 서열번호 30의 염기서열로 이루어진, 토마토 유래 APX4 유전자의 표적 염기서열에 특이적인 가이드 RNA와 엔도뉴클레아제 단백질의 복합체(ribonucleoprotein);를 이용하는 것일 수 있으나, 이에 제한되지 않는다.In the method for producing a genome-corrected tomato plant of the present invention, the introduction of the guide RNA and endonuclease protein of step (a) into tomato plant cells comprises the nucleotide sequence of SEQ ID NO: 30, tomato-derived APX4 gene a recombinant vector comprising DNA encoding a guide RNA specific for a target nucleotide sequence and a nucleic acid sequence encoding an endonuclease protein; Alternatively, a complex (ribonucleoprotein) of a guide RNA and an endonuclease protein specific to the target nucleotide sequence of the tomato-derived APX4 gene consisting of the nucleotide sequence of SEQ ID NO: 30 may be used, but is not limited thereto.
본 발명의 일 구현 예에 따른 유전체 교정 토마토 식물체의 제조방법에 있어서, 상기 서열번호 30의 염기서열은 다른 가이드 RNA 표적 서열인 서열번호 27 내지 서열번호 29의 염기서열에 비해 APX4 유전자의 표적 부위에서 InDel 돌연변이 유도 효율이 높은 것이 특징이다.In the method for producing a genome-corrected tomato plant according to an embodiment of the present invention, the nucleotide sequence of SEQ ID NO: 30 is compared to the nucleotide sequence of SEQ ID NO: 27 to SEQ ID NO: 29, which are other guide RNA target sequences, at the target site of the APX4 gene. It is characterized by high InDel mutagenesis efficiency.
본 발명에 따른 유전체 교정 토마토 식물체의 제조방법에 있어서, 상기 가이드 RNA와 엔도뉴클레아제 단백질의 복합체를 식물세포에 형질도입하는 방법은 전술한 것과 같다.In the method for producing a genome-corrected tomato plant according to the present invention, the method for transducing the complex of the guide RNA and the endonuclease protein into plant cells is the same as described above.
또한, 상기 표적 염기서열에 특이적인 가이드 RNA를 암호화하는 DNA 및 엔도뉴클레아제 단백질을 암호화하는 핵산 서열을 포함하는 재조합 벡터를 식물세포에 도입하는 것은 형질전환 방법을 의미한다. 식물 종의 형질전환은 이제는 쌍자엽 식물뿐만 아니라 단자엽 식물 양자를 포함한 식물 종에 대해 일반적이다. 원칙적으로, 임의의 형질전환 방법은 본 발명에 따른 재조합 벡터를 적당한 선조 세포로 도입시키는데 이용될 수 있다.In addition, introducing a recombinant vector including a DNA encoding a guide RNA specific for the target nucleotide sequence and a nucleic acid sequence encoding an endonuclease protein into a plant cell means a transformation method. Transformation of plant species is now common for plant species including both monocots as well as dicots. In principle, any transformation method can be used to introduce the recombinant vector according to the invention into suitable progenitor cells.
본 발명의 일 구현 예에 따른 유전체 교정 토마토 식물체의 제조방법에 있어서, 상기 재조합 벡터가 식물 세포에 형질전환되면, DNA 결합 및 절단 활성이 있는 엔도뉴클레아제 단백질과 상기 엔도뉴클레아제 단백질에 결합되며 표적 서열로 엔도뉴클레아제 단백질을 이끄는 sgRNA가 함께 발현되게 된다.In the method for producing a genome-corrected tomato plant according to an embodiment of the present invention, when the recombinant vector is transformed into a plant cell, it binds to an endonuclease protein having DNA binding and cleavage activity and the endonuclease protein and the sgRNA leading to the endonuclease protein as the target sequence is expressed together.
본 발명에 따른 유전체 교정 토마토 식물체의 제조방법에 있어서, 상기 표적 염기서열에 특이적인 가이드 RNA 및 엔도뉴클레아제 단백질이 도입되는 "식물세포"는 전술한 것과 같다.In the method for producing a genome-corrected tomato plant according to the present invention, "plant cells" into which the guide RNA and endonuclease protein specific to the target nucleotide sequence are introduced are the same as described above.
본 발명은 또한, 상기 유전체 교정 토마토 식물체의 제조방법에 의해 제조된 열매의 아스코르브산 함량이 증가된 유전체 교정 토마토 식물체 및 이의 유전체가 교정된 종자를 제공한다.The present invention also provides a genome-corrected tomato plant having an increased content of ascorbic acid in the fruit produced by the method for producing the genome-corrected tomato plant, and a seed whose genome is corrected.
본 발명에 따른 열매의 아스코르브산 함량이 증가된 유전체 교정 토마토 식물체는 토마토 열매에서 특이적으로 발현되는 APX4 (Ascorbate Peroxidase 4) 유전자를 CRISPR/Cas9 시스템을 이용하여 교정한 것으로, 토마토 유래 APX4 유전자가 녹-아웃되어, 유전체를 교정하지 않은 토마토 식물체에 비해 열매의 아스코르브산 함량이 증가된 형질을 가지는 유전체 교정 토마토 식물체이다.The genome-corrected tomato plant with an increased content of ascorbic acid in the fruit according to the present invention was obtained by correcting the APX4 (Ascorbate Peroxidase 4) gene specifically expressed in the tomato fruit using the CRISPR/Cas9 system, and the tomato-derived APX4 gene was -Out, it is a genome-corrected tomato plant having a trait that the ascorbic acid content of the fruit is increased compared to a tomato plant without genome editing.
이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail by way of Examples. However, the following examples only illustrate the present invention, and the content of the present invention is not limited to the following examples.
재료 및 방법Materials and Methods
1. 식물재료 및 배양조건1. Plant materials and culture conditions
본 발명에는 토마토 '마이크로톰' (Solanum lycopersicum cv. Micro-Tom) 품종을 사용하였다. 토마토 씨는 MS salt 4.4 g/L, 수크로스 30 g/L 및 plant agar 8 g/L 조성의 배지에서 발아시켰으며, 22~24℃, 16시간의 광(光), 8시간 암(暗) 상태로 유지되는 배양실에서 키웠다.In the present invention, tomato 'micro-tome' ( Solanum lycopersicum cv. Micro-Tom) variety was used. Tomato seeds were germinated in a medium composed of MS salt 4.4 g/L, sucrose 30 g/L, and plant agar 8 g/L, 22~24℃, 16 hours of light, 8 hours of dark. It was grown in a culture room maintained in a condition.
2. 2. SlAPXSlAPX 유전자 발현 패턴 분석 Gene expression pattern analysis
토마토의 SlAPX4SlAPX6 유전자의 발현 패턴을 비교 분석하기 위해 Sol Genomics Network의 Tomato Expression Database (TED) (http://ted.bti.cornell.edu/)와 'Heinz' 및 '마이크로톰' 토마토를 이용하였다. 유전자의 발현 패턴을 분석하기 위해 각 발달 단계에 해당하는 식물 시료를 채취한 뒤 액체질소를 이용하여 균질화 하였다. 이후 RNeasy Plant Mini Kit (Qiagen, Germany)를 사용하여 총 RNA를 추출하였다. 300 ng의 RNA를 대상으로 AMPIGENE cDNA Synthesis Kit (ENZO, USA)를 이용하여 cDNA를 제작한 후 Realtime PCR 분석을 실시하였다. Realtime PCR 분석은 1/10로 희석된 cDNA 4 ㎕, 멸균증류수 3 ㎕, 정방향 프라이머 2 ㎕, 역방향 프라이머 2 ㎕, SYBR Green (Qiagen, Germany) 10 ㎕를 넣은 후 95℃에서 15분 후, 95℃ 20초, 55℃ 30초, 72℃ 30초로 50 주기를 수행하였으며, 매 주기마다 형광신호를 검출하였다. 유전자 발현 분석에 사용한 프라이머 정보는 표 1에 기재하였다.To compare and analyze the expression patterns of SlAPX4 and SlAPX6 genes in tomato, Sol Genomics Network's Tomato Expression Database (TED) (http://ted.bti.cornell.edu/) and 'Heinz' and 'Microtome' tomatoes were used. . To analyze the gene expression pattern, plant samples corresponding to each developmental stage were collected and homogenized using liquid nitrogen. Then, total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen, Germany). For 300 ng of RNA, cDNA was prepared using the AMPIGENE cDNA Synthesis Kit (ENZO, USA), and then real-time PCR analysis was performed. Realtime PCR analysis was performed after adding 4 µl of cDNA diluted 1/10, 3 µl of sterile distilled water, 2 µl of forward primer, 2 µl of reverse primer, and 10 µl of SYBR Green (Qiagen, Germany) at 95°C for 15 minutes, 95°C 50 cycles were performed at 20 sec, 55 ℃ 30 sec, and 72 ℃ 30 sec, and the fluorescence signal was detected at every cycle. Primer information used for gene expression analysis is shown in Table 1.
Figure PCTKR2021019619-appb-img-000001
Figure PCTKR2021019619-appb-img-000001
3. 재조합 벡터(VIGS) 제작3. Construction of Recombinant Vector (VIGS)
토마토 SlAPX4SlAPX6 유전자의 발현 침묵을 유도하기 위해서 각 유전자마다 VIGS 시스템을 통해 침묵을 유도할 수 있는 표적 유전자 서열 세 지역을 선별한 뒤(표 2), 각 유전자를 합성하여 pUCIDT-Amp에 삽입하였다(cosmogenetech, Korea). 그 후 삽입된 유전자를 95℃에서 5분 후, 95℃ 30초, 57℃ 30초, 72℃ 20초로 25 주기를 수행하고 72℃에서 5분간 안정화시켰으며, PCR 산물을 아가로스 겔에 로딩하여 결과물을 확인하였다. 확인된 밴드를 Gel extraction kit (cosmogentech)를 사용하여 25 ㎕로 용출하여 insert 산물을 준비하였다. TRV2(pYL156)는 E.coli stock 5 ㎕을 카나마이신 50 ㎎/L이 함유된 LB 액체배지 (LB broth High Salt 25 g/L)에 접종하여 37℃에서 하루 동안 배양한 뒤, Nucleospin plasmid easypure kit (MACHEREY-NAGEL, Germany)를 통해 플라스미드를 추출하였다. 추출한 플라스미드는 EcoRI과 BamHI을 처리하여 37℃에서 하루 동안 반응시켜 절단시켰으며, 제한효소로 절단된 TRV2(pYL156) 벡터 4 ㎕, insert 4 ㎕, T4 ligase (cosmogentech), 10X 버퍼(cosmogentech) 1 ㎕, 멸균증류수 2 ㎕를 넣고 상온에서 2분 30초, 얼음에서 10분 처리하여 라이게이션(ligation)을 진행하여 재조합 벡터 [TRV2(pYL156)::SlAPX4 insert, TRV2(pYL156)::SlAPX6 insert]를 제작하였다(도 3). 제작된 재조합 벡터를 아그로박테리움 튜메파시엔스(Agrobacterium tumefaciens) GV3101에 삽입하였다. 유전자 존재 유무는 PCR과 염기서열 분석을 통해 확인하였으며, insert 합성 및 PCR 분석에 사용된 프라이머 정보는 상기 표 1에 기재하였다.To induce silencing of tomato SlAPX4 and SlAPX6 genes, three regions of target gene sequences capable of inducing silencing through the VIGS system for each gene were selected (Table 2), and each gene was synthesized and inserted into pUCIDT-Amp. (cosmogenetech, Korea). Then, 25 cycles of the inserted gene were performed at 95 ° C. for 5 minutes, 95 ° C. 30 seconds, 57 ° C. 30 seconds, 72 ° C. 20 seconds, and stabilized at 72 ° C. for 5 minutes, and the PCR product was loaded on an agarose gel. The result was confirmed. The identified band was eluted with 25 μl using a Gel extraction kit (cosmogentech) to prepare an insert product. TRV2(pYL156) was inoculated with 5 μl of E.coli stock in LB broth containing 50 mg/L of kanamycin (LB broth High Salt 25 g/L) and cultured for one day at 37°C, followed by Nucleospin plasmid easypure kit ( The plasmid was extracted through MACHEREY-NAGEL, Germany). The extracted plasmid was treated with EcoR I and BamHI and reacted for one day at 37° C. and cut, and restriction enzyme digested TRV2 (pYL156) vector 4 μl, insert 4 μl, T4 ligase (cosmogentech), 10X buffer (cosmogentech) 1 μl, 2 μl of sterile distilled water was added, and ligation was performed at room temperature for 2 minutes and 30 seconds and on ice for 10 minutes to proceed with ligation. ] was prepared (Fig. 3). The constructed recombinant vector was inserted into Agrobacterium tumefaciens GV3101. The presence or absence of a gene was confirmed through PCR and sequencing, and information on primers used for insert synthesis and PCR analysis is described in Table 1 above.
Figure PCTKR2021019619-appb-img-000002
Figure PCTKR2021019619-appb-img-000002
4. 토마토 식물체 VIGS 시스템 도입4. Introduction of the tomato plant VIGS system
재조합 벡터를 포함하고 있는 아그로박테리움 튜메파시엔스(A. tumefaciens) GV3101을 리팜피신 50 ㎎/L, 카나마이신 50 ㎎/L이 함유된 YEP 액체배지 (yeast extract 10 g/L, peptone 10 g/L, NaCI 5 g/L, pH 7.2)에서 하루 동안 배양시킨다. 배양된 균액 0.5 ㎖에 리팜피신 50 ㎎/L, 카나마이신 50 ㎎/L가 포함된 YEP 액체 배지 5 ㎖을 넣어준 뒤, O.D600 값이 0.8~1.0이 될 때까지 배양하고 원심분리하여 균주를 포집하였다. 그 후 상기 균주를 계대했던 배지와 1:1의 비율로 10 mM MgCl2를 첨가하여 재부유시킨 후, MgCl2를 첨가하며 OD600에서 1.0을 맞춰주었다. 200 μM 아세토시린곤(acetosyringone)와 10 mM MES를 첨가 후 상온에서 4시간 동안 암배양하였다. 그 후 토마토 mature green 단계의 열매에 균액을 주입하였다.Agrobacterium tumefaciens containing the recombinant vector ( A. tumefaciens ) GV3101 rifampicin 50 mg / L, kanamycin 50 mg / L YEP liquid medium (yeast extract 10 g / L, peptone 10 g / L, NaCI 5 g/L, pH 7.2) for one day. After adding 5 ml of YEP liquid medium containing 50 mg/L of rifampicin and 50 mg/L of kanamycin to 0.5 ml of the cultured bacterial solution, cultured until the OD 600 value reached 0.8-1.0, and centrifuged to collect the strain. . Thereafter, the strain was resuspended by adding 10 mM MgCl 2 at a ratio of 1:1 with the medium in which the strain was passaged, and then MgCl 2 was added to adjust the OD to 1.0 at 600 . After the addition of 200 μM acetosyringone and 10 mM MES, dark culture was performed at room temperature for 4 hours. After that, the fungus was injected into the fruits of the tomato mature green stage.
5. CRISPR/Cas9 벡터 제작5. CRISPR/Cas9 vector construction
SlAPX4를 녹아웃(Knock-out)하기 위하여,SlAPX4 유전자 서열을 대상으로 CRISPR/Cas9 시스템을 적용할 수 있는 sgRNA를 제작하고 Golden Gate system을 이용하여 벡터(pAGM4723::NPTⅡ::Cas9::sgRNA1-4::L3E)를 제작한 뒤, 아그로박테리움 튜메파시엔스 LBA4404에 삽입하였다. 유전자 존재 유무는 표 4에 기재된 프라이머를 이용한 PCR과 염기서열 분석을 통해 확인하였다.In order to knock-out SlAPX4 , sgRNA to which the CRISPR/Cas9 system can be applied to the SlAPX4 gene sequence was prepared and the vector (pAGM4723::NPTII::Cas9::sgRNA1-4 using the Golden Gate system) ::L3E) was prepared and then inserted into Agrobacterium tumefaciens LBA4404. The presence or absence of the gene was confirmed through PCR and nucleotide sequence analysis using the primers shown in Table 4.
6. CRISPR/Cas9 시스템을 이용한 토마토 형질전환체 개발6. Tomato transformant development using CRISPR/Cas9 system
CRISPR/Cas9 벡터를 포함하고 있는 아그로박테리움 튜메파시엔스 LBA4404 균주를 YEP 액체배지 (yeast extract 10 g/L, peptone 10 g/L, NaCI 5 g/L, pH 7.2)에서 O.D600 값이 0.7~0.8이 될 때까지 배양한 후,원심분리하여 균주를 포집하였다. 그 후 상기 균주를 1/2 MS 배지 (MS salt 2.2 g/L, sucrose 20 g/L, MES 0.5 g/L, pH 5.7)와 200 μM 아세토시린곤(acetosyringone)을 넣고 재부유시킨 후,28℃에서 4시간 동안 배양하였다. 그 후, 상기 배양액을 2 등분하여 잘라놓은 토마토의 떡잎 조각과 10분간 공동 배양한 후, 떡잎을 MT-COM (co-culture medium)에 잎의 기공이 아래로 향하도록 배열한 뒤,배양용기를 밀봉하여 2일 동안 암배양하였다. 2일 후에 MT-COM 배지의 토마토 떡잎 조각들을 잎의 기공이 위로 향하도록 하여 MT-SRM (shoot induction medium)에 옮겨주었다. 2주에 한번씩 배지를 바꿔주며 상태를 관찰한 뒤, 신초(shoot)가 유도되면 MT-SEM (shoot elongation medium)에 옮겨주어 완전한 줄기로 분화시켰다. 줄기가 완전히 형성되면 식물체를 MT-RM (root induction medium)로 옮겨 뿌리를 유도하였다. 뿌리까지 완전하게 유도된 식물체는 흙으로 옮겨 생장시켰다. 각 실험에 사용된 배지의 조성은 하기 표 3에 나타내었다.The Agrobacterium tumefaciens LBA4404 strain containing the CRISPR/Cas9 vector had an OD 600 value of 0.7~ After culturing until 0.8, the strain was collected by centrifugation. After that, the strain was resuspended in 1/2 MS medium (MS salt 2.2 g/L, sucrose 20 g/L, MES 0.5 g/L, pH 5.7) and 200 μM acetosyringone and resuspended, 28 Incubated at ℃ for 4 hours. After that, the culture solution was divided into two and co-cultured with the cotyledon leaf pieces of the cut tomato for 10 minutes, and then the cotyledon leaves were arranged in MT-COM (co-culture medium) so that the pores of the leaves face down, and the culture vessel was It was sealed and darkly cultured for 2 days. Two days later, tomato cotyledon pieces in MT-COM medium were transferred to MT-SRM (shoot induction medium) with the stomata of the leaves facing upward. After changing the medium once every 2 weeks and observing the condition, when shoots were induced, they were transferred to MT-SEM (shoot elongation medium) and differentiated into complete stems. When the stem was completely formed, the plant was transferred to MT-RM (root induction medium) to induce roots. Plants completely induced to the roots were transferred to soil and grown. The composition of the medium used in each experiment is shown in Table 3 below.
조직 배양 배지 조성Tissue culture medium composition
공배양 배지
(Co-culture medium)
co-culture medium
(Co-culture medium)
신초유도배지(Shoot
induction medium)
New colostrum induction medium (Shoot)
induction medium)
신초신장배지(Shoot
elongation medium)
New growth medium (Shoot)
elongation medium)
발근유도배지(Root
induction medium)
Root induction medium (Root)
induction medium)
15 g/L sucrose 15 g/L sucrose 30 g/L sucrose30 g/L sucrose 15 g/L sucrose15 g/L sucrose 15 g/L sucrose15 g/L sucrose
4.4 g/L MS salt4.4 g/L MS salt 4.4 g/L MS salt4.4 g/L MS salt 4.4 g/L MS salt4.4 g/L MS salt 4.4 g/L MS salt4.4 g/L MS salt
1 μM IAA1 μM IAA 50 ㎎/L kanamycin50 mg/L kanamycin 50 ㎎/L kanamycin50 mg/L kanamycin 50 ㎎/L kanamycin50 mg/L kanamycin
10 μM Zeatin10 μM Zeatin 0.1㎎/L IAA0.1mg/L IAA 250 ㎎/L Cefotaxim250 mg/L Cefotaxim 2 ㎎/L IBA2 mg/L IBA
200 μM acetosyringone200 μM acetosyringone 2 ㎎/L Zeatin2 mg/L Zeatin 8 g/L plant agar8 g/L plant agar 250 ㎎/L Cefotaxim250 mg/L Cefotaxim
8 g/L plant agar8 g/L plant agar 250 ㎎/L Cefotaxtim250 mg/L Cefotaxtim 8 g/L plant agar8 g/L plant agar
8 g/L plant agar8 g/L plant agar
(pH 6.0)(pH 6.0) (pH 6.0)(pH 6.0) (pH 6.0)(pH 6.0) (pH 6.0)(pH 6.0)
7. CRISPR/Cas 9 시스템에 의한 표적 부위 돌연변이 확인7. Identification of target site mutations by the CRISPR/Cas 9 system
형질전환 식물체에서 CRISPR/Cas 9 시스템에 의해 표적 부위에 돌연변이가 유도되었는지를 확인하기 위해 표적 부위 주변의 약 600 bp 정도를 PCR로 증폭하였다. 형질전환체 확인 및 표적부위 돌연변이 확인을 위한 PCR 프라이머 정보는 표 4에 나타내었다. 그 후 염기서열 분석을 통해 표적 부위의 염기서열 변화를 확인하여 CRISPR/Cas9 시스템에 의해 표적 부위에 돌연변이 여부를 판단하였다.In the transgenic plant, about 600 bp around the target site was amplified by PCR to confirm whether the mutation was induced in the target site by the CRISPR/Cas 9 system. PCR primer information for transformant confirmation and target site mutation confirmation is shown in Table 4. Thereafter, the change in the nucleotide sequence of the target site was confirmed through sequencing, and it was determined whether the target site was mutated by the CRISPR/Cas9 system.
CRISPR/Cas 9 시스템을 이용한 형질전환체의 확인 및 표적부위 돌연변이 확인을 위한 PCR 프라이머 정보PCR primer information for identification of transformants and target site mutations using the CRISPR/Cas 9 system
프라이머 명칭Primer name 염기서열 (5'→3')base sequence (5'→3') 용도purpose
NPTⅡ-FNPTⅡ-F AACAAGATGGATTGCACGCA (서열번호 31)AACAAGATGGATTGCACGCA (SEQ ID NO: 31) Transgenic plant screeningtransgenic plant screening
NPTⅡ-RNPTⅡ-R AAGAAGGCGATAGAAGGCGA (서열번호 32)AAGAAGGCGATAGAAGGCGA (SEQ ID NO: 32)
Cas9-300end-FCas9-300end-F GACGCTAACCTCGATAAGGT (서열번호 33)GACGCTAACCTCGATAAGGT (SEQ ID NO: 33)
NOS-RNOS-R CAAGACCGGCAACAGGATTCAATC (서열번호 34)CAAGACCGGCAACAGGATTCAATC (SEQ ID NO: 34)
APX Set 2-FAPX Set 2-F CTTGGTACTAGTCGTAATGTG (서열번호 35)CTTGGTACTAGTCGTAATGTG (SEQ ID NO: 35) Amplification of the target regionAmplification of the target region
APX Set 2-RAPX Set 2-R GAAACCATGTATTGCATCTTC (서열번호 36)GAAACCATGTATTGCATCTTC (SEQ ID NO: 36)
8. 아스코르브산 분석8. Ascorbic Acid Assay
열매에서 아스코르브산 측정은 Abcam사의 Ascorbic Acid Assay Kit를 이용하여 측정되었다. 야생형과 VIGS 시스템을 통해 유전자 침묵을 유도시킨 식물체 또는 CRISPR/Cas 9 시스템에 의한 형질전환 식물체의 ripen red 상태 열매를 실험에 이용하였다. 열매 조직 40 mg을 PBS 용액으로 세척한 후 400 ㎕의 멸균증류수와 함께 튜브에 넣어 균질기를 이용하여 균질화시킨 후 샘플을 4℃에서 13,000 rpm으로 5분간 원심분리한 후 상등액을 취해 제조사의 분석법에 따라 분광광도계(microplate reader)를 이용하여 OD593에서 각 샘플의 아스코르브산 함량을 측정하였다.Ascorbic acid was measured in the fruit using Abcam's Ascorbic Acid Assay Kit. Wild-type and ripen red fruits of plants inducing gene silencing through the VIGS system or transgenic plants by the CRISPR/Cas 9 system were used for the experiment. After washing 40 mg of fruit tissue with PBS solution, put it in a tube with 400 μl of sterile distilled water and homogenize it using a homogenizer. After centrifuging the sample at 4° C. at 13,000 rpm for 5 minutes, take the supernatant and take the supernatant according to the manufacturer’s analysis method. The ascorbic acid content of each sample was measured at OD 593 using a spectrophotometer (microplate reader).
실시예 1. 토마토에서 Example 1. In tomatoes SlAPXSlAPX 유전자 발현양상 확인 Confirmation of gene expression pattern
본 발명자들은 토마토에 존재하는 SlAPX 유전자 중 토마토 열매에서 아스코르브산 함량 조절에 있어 SlAPX4 (Solyc09g007270) 유전자의 기능을 분석하기 위하여 기존에 토마토에서 아스코르브산 함량 조절에 관여하는 것으로 알려져 있는 SlAPX6 (Solyc11g01855) 유전자와의 비교 분석을 실시하였다.The present inventors analyzed the function of the SlAPX4 ( Solyc09g007270 ) gene in regulating the ascorbic acid content in tomato fruits among the SlAPX genes present in tomatoes . A comparative analysis was performed.
유전자 서열 분석을 통해 SlAPX4SlAPX6는 서열 유사도가 높지 않은 유전자임을 확인하였다(도 1). 그 후, SlAPX4SlAPX6의 발현 패턴을 Heinz 품종과 마이크로톰 품종을 대상으로 분석하였다. Sol Genomics Network의 Tomato Expression Database (TED)를 통해 Heinz에서 SlAPX4는 토마토 열매에서 발현되며, 특히 열매 발달 후반부에 발현이 크게 증가하는 것으로 나타났다(도 2A). SlAPX4와 달리 SlAPX6는 잎 조직에서 가장 높은 발현 패턴을 보였으며, 토마토 열매에서는 Immature green 단계에서 가장 높은 발현을 보인 후 점차 감소하는 패턴을 보였다(도 2A). 상기와 같은 발현 패턴은 토마토 마이크로톰 품종에서도 유사하게 관찰되었다(도 2B). 또한 두 유전자의 발현 패턴을 비교 분석한 결과 SlAPX4SlAPX6에 비해 열매 조직에서 보다 높은 발현량을 보였음을 알 수 있었다(도 2C). 이상의 결과를 통해 SlAPX4는 열매 발달 후반부에 발현이 증가하는 유전자이고 SlAPX6는 잎에서 높게 발현되며 열매 발달 단계에서는 발현이 낮은 유전자임을 확인하였다.Through gene sequence analysis, it was confirmed that SlAPX4 and SlAPX6 were genes with low sequence similarity (FIG. 1). Then, the expression patterns of SlAPX4 and SlAPX6 were analyzed for Heinz and Microtome varieties. Sol Genomics Network's Tomato Expression Database (TED) showed that SlAPX4 in Heinz was expressed in tomato fruits, and, in particular, its expression significantly increased in the late stages of fruit development (FIG. 2A). Unlike SlAPX4 , SlAPX6 showed the highest expression pattern in leaf tissue, and showed the highest expression pattern in the immature green stage in tomato fruit, and then showed a gradually decreasing pattern (FIG. 2A). The above expression pattern was similarly observed in the tomato microtome variety (FIG. 2B). In addition, as a result of comparative analysis of the expression patterns of the two genes, it was found that SlAPX4 showed a higher expression level in fruit tissues than SlAPX6 (FIG. 2C). From the above results, it was confirmed that SlAPX4 is a gene whose expression is increased in the late stage of fruit development, while SlAPX6 is a gene that is highly expressed in leaves and low in expression in the stage of fruit development.
실시예 2. Example 2. SlAPX4SlAPX4 또는 or SlAPX6SlAPX6 유전자 침묵에 의한 아스코르브산 함량 변화 Changes in ascorbic acid content by gene silencing
토마토 열매의 아스코르브산 함량 조절에 있어 SlAPX4SlAPX6 유전자의 기능을 비교·분석하기 위하여 각 유전자의 발현을 침묵시킬 수 있는 VIGS 시스템을 도입하였다. SlAPX4SlAPX6 유전자의 발현을 침묵시키기 위해 유전자의 코딩 서열(coding sequence)의 세 지역을 대상으로 VIGS 벡터 (TRV1, TRV2:insert)를 제작하였다(도 3). 각 벡터의 insert는 SlAPX4SlAPX6 유전자의 서열 중 250~300 bp의 서열로 상세 서열은 표 2에 제시하였다. 제작한 재조합 벡터는 아그로박테리움 GV3103을 매개로 mature green 단계의 토마토 열매에 주입하였다. 감염 후 3주 경과 후 Ripen Red 시기의 열매를 수확하여 열매 내 아스코르브산 함량을 분석한 결과 SlAPX4 유전자를 침묵시켰을 때, 대조군에 비해 아스코르브산 함량이 높게 나타난 것을 확인하였다(도 4A). 이에 비해 SlAPX6 유전자를 침묵시켰을 경우, 대조군에 비해 아스코르브산 함량이 증가하지 않고 오히려 감소하였다(도 4B). 이 결과들을 토대로 SlAPX4 유전자가 토마토 열매에서 특이적으로 아스코르브산 함량을 조절함을 규명하였고,SlAPX4 유전자 침묵을 통해 토마토 열매 내 아스코르브산 함량 증가를 유도할 수 있음을 확인하였다.In order to compare and analyze the functions of SlAPX4 and SlAPX6 genes in controlling the content of ascorbic acid in tomato fruit, a VIGS system that can silence the expression of each gene was introduced. To silence the expression of SlAPX4 and SlAPX6 genes, VIGS vectors (TRV1, TRV2:insert) were constructed for three regions of the gene coding sequence (FIG. 3). The insert of each vector is a sequence of 250-300 bp among the sequences of the SlAPX4 and SlAPX6 genes, and detailed sequences are shown in Table 2. The prepared recombinant vector was injected into mature green tomatoes through Agrobacterium GV3103. Three weeks after infection, the fruits of the Ripen Red period were harvested and the ascorbic acid content in the fruits was analyzed. As a result, when the SlAPX4 gene was silenced, it was confirmed that the ascorbic acid content was higher than that of the control group (FIG. 4A). In contrast, when the SlAPX6 gene was silenced, the ascorbic acid content did not increase, but rather decreased compared to the control group (FIG. 4B). Based on these results, it was confirmed that the SlAPX4 gene specifically regulates the ascorbic acid content in tomato fruits, and it was confirmed that the SlAPX4 gene silencing could induce an increase in the ascorbic acid content in the tomato fruits.
실시예 3. sgRNA의 위치에 따른 CRISPR/Cas9 시스템 효율 비교Example 3. Comparison of CRISPR/Cas9 system efficiency according to the location of sgRNA
SlAPX4 유전자의 게놈 DNA 서열(서열번호 38)에서 sgRNA로 사용가능한 서열을 선발하여 4개의 sgRNA를 제작하였다. 본 발명에 있어서, 서열번호 27 내지 30의 염기서열은 하기 표 5의 표적 서열에서 PAM 염기(밑줄 표시)를 제외한 서열을 지칭한다.Four sgRNAs were prepared by selecting sequences usable as sgRNAs from the genomic DNA sequence (SEQ ID NO: 38) of the SlAPX4 gene. In the present invention, the nucleotide sequences of SEQ ID NOs: 27 to 30 refer to sequences excluding the PAM base (underlined) from the target sequence of Table 5 below.
SlAPX4 교정을 위한 sgRNA 후보 서열 sgRNA candidate sequence for SlAPX4 correction
표적 서열 (23bp/with PAM)target sequence (23bp/with PAM )
gRNA1gRNA1 TTTGGAAATCGACGTTTGATCGG (서열번호 27)TTTGGAAATCGACGTTTGAT CGG (SEQ ID NO: 27)
gRNA2gRNA2 AAGCAGTTGAAAAATGTAAGAGG (서열번호 28)AAGCAGTTGAAAAATGTAAG AGG (SEQ ID NO: 28)
gRNA3gRNA3 GTTAAGAATTTTTTACATGATGG (서열번호 29)GTTAAGAATTTTTTACATGA TGG (SEQ ID NO: 29)
gRNA4gRNA4 GATGTCAAAACCAAAACTGGTGG (서열번호 30)GATGTCAAAACCAAAACTGG TGG (SEQ ID NO: 30)
그 후 각각의 sgRNA와 CRISPR/Cas9 시스템을 가진 벡터를 Golden Gate system (Engler C et al., ACS Synth Biol. 2014, 3(11):839-43)을 이용하여 제작한 후, 아그로박테리움 튜메파시엔스 LBA4404에 형질전환시켜 토마토 떡잎에 Agro-infiltration 방법을 통해 CRISPR/Cas9 시스템을 일시적으로 발현시켰다. 그 후 DNA를 추출하여 pGEM-T Easy 벡터(Promega, USA)에 클로닝한 뒤, 콜로니의 염기서열을 확인하는 방법을 통해 CRISPR/Cas9 매개 돌연변이가 일어났는지를 확인하였다. 콜로니는 얻어지는 개수만큼 모두 PCR을 수행하여 염기서열을 분석하였으며, 효율은 다음의 식을 이용하여 비교하였다.After that, each sgRNA and a vector having a CRISPR/Cas9 system were prepared using the Golden Gate system (Engler C et al., ACS Synth Biol. 2014, 3(11):839-43), and then Agrobacterium tume After transformation into Fasiens LBA4404, the CRISPR/Cas9 system was transiently expressed in tomato cotyledons through the Agro-infiltration method. After DNA was extracted and cloned into pGEM-T Easy vector (Promega, USA), it was confirmed whether CRISPR/Cas9 mediated mutation occurred by confirming the nucleotide sequence of the colony. As many colonies were obtained, PCR was performed to analyze the nucleotide sequence, and the efficiency was compared using the following formula.
Efficiency(%) = (number of mutated colonies/number of total colonies) x 100Efficiency (%) = (number of mutated colonies/number of total colonies) x 100
CRISPR/Cas9 매개 돌연변이는 염기서열 분석 원시데이터 중 CRISPR/Cas9 system 편집 위치인 PAM 서열 +3 bp 앞에서의 변화를 야생형의 염기서열과 비교하는 방법을 통해 확인하였고, 염기 삽입 또는 결실, 이중 피크 등이 일어난 것을 돌연변이가 일어난 것으로 판단하였다. 그 결과, sgRNA1은 총 442개의 콜로니를 얻어 염기서열 분석을 진행한 결과, PAM 서열 +3 bp 앞에서 아무런 변화가 나타나지 않았고, 이는 sgRNA2의 273개 콜로니에서도 마찬가지였다. sgRNA3의 경우 총 548개의 콜로니를 분석한 결과 2개에서 표적 유전자 부위에 돌연변이가 관찰되었고, sgRNA4의 경우 총 561개의 콜로니를 분석한 결과 5개의 콜로니에서 PAM 부분 +3 bp 서열에서 삽입 또는 결실이 관찰되어 CRISPR/Cas9 매개 돌연변이가 일어났음을 확인할 수 있었다. 즉, SlAPX4에 대한 각 sgRNA의 돌연변이 효율은 sgRNA1 및 sgRNA2가 0%, sgRNA3이 0.36%, sgRNA4가 0.89%로, 그 중 효율이 높은 sgRNA4를 이용하여 식물 형질전환을 진행하였다.CRISPR/Cas9-mediated mutation was confirmed by comparing the change in front of the PAM sequence +3 bp, which is the CRISPR/Cas9 system editing site, with the wild-type nucleotide sequence in the nucleotide sequence analysis raw data, and there were no nucleotide insertions or deletions, double peaks, etc. It was judged that a mutation had occurred. As a result, a total of 442 colonies of sgRNA1 were obtained and sequencing was performed. As a result, no change was observed in front of the PAM sequence +3 bp, which was also the case in 273 colonies of sgRNA2. In the case of sgRNA3, when a total of 548 colonies were analyzed, mutations were observed in two of the target gene sites, and in the case of sgRNA4, as a result of analyzing a total of 561 colonies, an insertion or deletion was observed in the PAM part +3 bp sequence in 5 colonies. It was confirmed that CRISPR/Cas9-mediated mutation occurred. That is, the mutation efficiency of each sgRNA for SlAPX4 was 0% for sgRNA1 and sgRNA2, 0.36% for sgRNA3, and 0.89% for sgRNA4. Among them, sgRNA4 with high efficiency was used for plant transformation.
실시예 4. Example 4. SlAPX4SlAPX4 유전자교정을 위한 형질전환 토마토 개발 및 목표 형질 세대 고정 확인 Development of transgenic tomato for gene editing and confirmation of target trait generation fixation
CRISPR/Cas9 시스템을 가진 pAGM4723::NPTⅡ::Cas9::sgRNA4::L3E 재조합벡터(도 5B)가 도입된 LBA4404 균주를 이용하여 아그로박테리움 매개 형질전환 방법으로 토마토 형질전환을 수행하였다. 아그로박테리움과 공배양한 토마토 조직을 카나마이신이 포함된 배지에서 재분화시켜 1차적으로 형질전환 식물체를 선별하였으며, 식물체 잎에서 genomic DNA를 추출하여 Cas9과 NPTⅡ가 모두 발현된 식물체를 선발하여 형질전환 토마토를 개발하였다.Tomato transformation was performed by an Agrobacterium-mediated transformation method using the LBA4404 strain into which the pAGM4723::NPTII::Cas9::sgRNA4::L3E recombinant vector (FIG. 5B) with CRISPR/Cas9 system was introduced. Transgenic plants were primarily selected by redifferentiating tomato tissues co-cultured with Agrobacterium in a medium containing kanamycin, and by extracting genomic DNA from plant leaves, plants expressing both Cas9 and NPTII were selected and transformed tomatoes has been developed.
선발된 형질전환 토마토는 모두 APX set2-F, APX set2-R 프라이머를 이용하여 표적 유전자 주변의 600 bp 정도를 PCR로 증폭한 후 염기서열 분석을 진행하여 CRISPR/Cas9 매개 돌연변이를 확인하였다. 염기서열 분석 결과 CRISPR/Cas9 system 편집 위치인 PAM 서열 앞 +3 bp에서 CRISPR/Cas9 매개 돌연변이가 일어난 식물체들을 선발하였다. SlAPX4 유전자교정이 일어난 형질전환 토마토의 종자를 수확하여 2세대 분석을 실시하였다. 종자를 흙에 파종하여 발아시킨 뒤 충분히 자란 식물체의 잎에서 genomic DNA를 추출하여 표적 유전자 부근 서열을 PCR과 염기서열 분석을 통해 유전자 편집여부를 확인하였다(도 6A). SlAPX4 유전자에 염기 결실 또는 삽입에 의해 frame-shift 돌연변이가 생긴 유전자교정체에서 ripen red 시기의 열매 내 아스코르브산 함량을 분석한 결과 유전자교정이 일어난 2세대 유전자교정 토마토에서 아스코르브산의 함량이 대조군에 비해 두배 이상 높게 나타난 것을 확인하였다(도 6B). 이 결과들을 토대로 SlAPX4 유전자가 토마토 열매의 아스코르브산 함량을 조절함을 규명하였고,SlAPX4 유전자교정이 토마토 열매 내 아스코르브산 함량 증가를 유도함을 확인하였다. 본 과정을 통해 SlAPX4 유전자 서열에 돌연변이가 존재하는 아스코르브산의 함량이 증가된 유전자교정 토마토를 개발하였다.All of the selected transformed tomatoes were amplified by PCR to about 600 bp around the target gene using APX set2-F and APX set2-R primers, and then sequencing was performed to confirm CRISPR/Cas9 mediated mutations. As a result of sequencing, plants in which CRISPR/Cas9 mediated mutation occurred at +3 bp before the PAM sequence, which is the CRISPR/Cas9 system editing site, were selected. The second generation analysis was performed by harvesting the seeds of the transgenic tomato in which SlAPX4 gene editing occurred. After the seeds were sown in soil and germinated, genomic DNA was extracted from the leaves of sufficiently grown plants, and the sequence near the target gene was checked for gene editing through PCR and sequencing (FIG. 6A). As a result of analyzing the ascorbic acid content in the ripen red fruit in the modified gene in which the frame-shift mutation was caused by nucleotide deletion or insertion in the SlAPX4 gene, the content of ascorbic acid in the second-generation gene-edited tomato with gene editing was higher than that of the control group. It was confirmed that it appeared more than twice as high (FIG. 6B). Based on these results, it was confirmed that the SlAPX4 gene regulates the ascorbic acid content of tomato fruits, and it was confirmed that the SlAPX4 gene editing induces an increase in the ascorbic acid content in tomato fruits. Through this process, a gene-edited tomato with an increased content of ascorbic acid having a mutation in the SlAPX4 gene sequence was developed.

Claims (14)

  1. 서열번호 2의 아미노산 서열로 이루어진 토마토(Solanum lycopersicum) 유래 APX4 (Ascorbate Peroxidase 4) 단백질 코딩 유전자의 발현을 조절하는 단계를 포함하는, 토마토 열매의 아스코르브산 함량 조절 방법.Tomato ( Solanum lycopersicum ) consisting of the amino acid sequence of SEQ ID NO: 2 APX4 (Ascorbate Peroxidase 4) A method of regulating the content of ascorbic acid in tomato fruit, comprising regulating the expression of a protein-coding gene.
  2. 제1항에 있어서, 상기 토마토 유래 APX4 단백질 코딩 유전자의 발현 조절은 APX4 유전자의 발현을 저해하여 아스코르브산 함량을 증가시키는 것을 특징으로 하는 토마토 열매의 아스코르브산 함량 조절 방법.The method according to claim 1, wherein the control of the expression of the tomato-derived APX4 protein-coding gene inhibits the expression of the APX4 gene to increase the ascorbic acid content of tomato fruits.
  3. 제2항에 있어서, 상기 APX4 유전자의 발현 저해는 VIGS (Virus-induced gene silencing), RNAi 또는 안티센스 RNA, T-DNA 삽입, 내생 트랜스포존(transposon), 방사선 조사 또는 유전자 교정 시스템을 통한 돌연변이 유발을 이용하여 발현을 저해하는 것을 특징으로 하는 토마토 열매의 아스코르브산 함량 조절 방법.According to claim 2, wherein the inhibition of the expression of the APX4 gene VIGS (Virus-induced gene silencing), RNAi or antisense RNA, T-DNA insertion, endogenous transposon (transposon), irradiation or using mutagenesis through a gene editing system A method for controlling the content of ascorbic acid in tomato fruit, characterized in that it inhibits expression.
  4. (a) 토마토(Solanum lycopersicum) 유래 APX4 (Ascorbate Peroxidase 4) 단백질 코딩 유전자를 포함하는 재조합 벡터로 토마토 식물세포를 형질전환하는 단계; 및(A) tomato ( Solanum lycopersicum ) transforming tomato plant cells with a recombinant vector containing a protein-coding gene APX4 (Ascorbate Peroxidase 4) derived from; and
    (b) 상기 형질전환된 토마토 식물세포로부터 형질전환된 토마토 식물체를 재분화하는 단계;를 포함하는 열매의 아스코르브산 함량이 조절된 형질전환 토마토 식물체의 제조방법.(b) re-differentiating the transformed tomato plant from the transformed tomato plant cells; a method for producing a transformed tomato plant in which the ascorbic acid content of the fruit is controlled, comprising a.
  5. 제4항에 있어서, 상기 토마토 유래 APX4 단백질 코딩 유전자의 발현을 저해시켜 야생형에 비해 열매의 아스코르브산 함량이 증가된 것을 특징으로 하는 형질전환 토마토 식물체의 제조방법.[Claim 5] The method for producing a transgenic tomato plant according to claim 4, wherein the tomato-derived APX4 protein coding gene is inhibited to increase the ascorbic acid content of the fruit compared to the wild type.
  6. 제4항 또는 제5항의 방법에 의해 제조된 열매의 아스코르브산 함량이 증가된 형질전환 토마토 식물체.A transgenic tomato plant having an increased content of ascorbic acid in the fruit produced by the method of claim 4 or 5.
  7. 제6항에 따른 식물체의 형질전환된 종자.The transformed seed of the plant according to claim 6 .
  8. 서열번호 2의 아미노산 서열로 이루어진 토마토(Solanum lycopersicum) 유래 APX4 (Ascorbate Peroxidase 4) 단백질을 코딩하는 유전자를 유효성분으로 함유하는 토마토 열매의 아스코르브산 함량 조절용 조성물.A composition for regulating the content of ascorbic acid in tomato fruits containing a gene encoding APX4 (Ascorbate Peroxidase 4) protein derived from tomato ( Solanum lycopersicum ) consisting of the amino acid sequence of SEQ ID NO: 2 as an active ingredient.
  9. 서열번호 30의 염기서열로 이루어진, 토마토(Solanum lycopersicum) 유래 APX4 (Ascorbate Peroxidase 4) 유전자의 표적 염기서열에 특이적인 가이드 RNA(guide RNA)를 암호화하는 DNA 및 엔도뉴클레아제(endonuclease) 단백질을 암호화하는 핵산 서열을 포함하는 재조합 벡터; 또는 서열번호 30의 염기서열로 이루어진, 토마토 유래 APX4 유전자의 표적 염기서열에 특이적인 가이드 RNA와 엔도뉴클레아제 단백질의 복합체(ribonucleoprotein);를 유효성분으로 함유하는, 토마토 열매의 아스코르브산 함량을 증가시키기 위한 유전체 교정용 조성물.Consisting of the nucleotide sequence of SEQ ID NO: 30, tomato ( Solanum lycopersicum )-derived APX4 (Ascorbate Peroxidase 4) DNA encoding a specific guide RNA (guide RNA) to the target nucleotide sequence of the gene and endonuclease (endonuclease) encoding the protein a recombinant vector comprising a nucleic acid sequence; or a complex (ribonucleoprotein) of a guide RNA and endonuclease protein specific for the target nucleotide sequence of the tomato-derived APX4 gene, consisting of the nucleotide sequence of SEQ ID NO: 30; as an active ingredient, increase the ascorbic acid content of tomato fruits A composition for genome editing for
  10. 서열번호 30의 염기서열로 이루어진, 토마토(Solanum lycopersicum) 유래 APX4 (Ascorbate Peroxidase 4) 유전자의 표적 염기서열에 특이적인 가이드 RNA를 유효성분으로 함유하는, 토마토 열매의 아스코르브산 함량을 증가시키기 위한 유전체 교정용 조성물.The nucleotide sequence of SEQ ID NO: 30, comprising a guide RNA specific to the target nucleotide sequence of APX4 (Ascorbate Peroxidase 4) gene derived from tomato ( Solanum lycopersicum ) as an active ingredient, genome editing to increase the ascorbic acid content of tomato fruits for composition.
  11. (a) 서열번호 30의 염기서열로 이루어진, 토마토(Solanum lycopersicum) 유래 APX4 (Ascorbate Peroxidase 4) 유전자의 표적 염기서열에 특이적인 가이드 RNA(guide RNA) 및 엔도뉴클레아제(endonuclease) 단백질을 토마토 식물세포에 도입하여 유전체를 교정하는 단계; 및(a) tomato ( Solanum lycopersicum ) consisting of the nucleotide sequence of SEQ ID NO: 30, a guide RNA (guide RNA) and endonuclease (endonuclease) protein specific to the target nucleotide sequence of APX4 (Ascorbate Peroxidase 4) gene derived from tomato plant Correcting the genome by introducing it into the cell; and
    (b) 상기 유전체가 교정된 토마토 식물세포로부터 토마토 식물체를 재분화하는 단계;를 포함하는, 열매의 아스코르브산 함량이 증가된 유전체 교정 토마토 식물체의 제조방법.(b) redifferentiating the tomato plant from the tomato plant cell in which the genome has been corrected; comprising, a method for producing a genome-corrected tomato plant with an increased content of ascorbic acid in the fruit.
  12. 제11항에 있어서, 상기 (a) 단계의 가이드 RNA 및 엔도뉴클레아제 단백질을 토마토 식물세포에 도입하는 것은, 서열번호 30의 염기서열로 이루어진, 토마토 유래 APX4 유전자의 표적 염기서열에 특이적인 가이드 RNA를 암호화하는 DNA 및 엔도뉴클레아제 단백질을 암호화하는 핵산 서열을 포함하는 재조합 벡터; 또는 서열번호 30의 염기서열로 이루어진, 토마토 유래 APX4 유전자의 표적 염기서열에 특이적인 가이드 RNA와 엔도뉴클레아제 단백질의 복합체(ribonucleoprotein);를 이용하는 것을 특징으로 하는 제조방법.12. The method of claim 11, wherein the introduction of the guide RNA and endonuclease protein in step (a) into tomato plant cells consists of the nucleotide sequence of SEQ ID NO: 30, a guide specific to the target nucleotide sequence of the tomato-derived APX4 gene a recombinant vector comprising a DNA encoding RNA and a nucleic acid sequence encoding an endonuclease protein; Or the nucleotide sequence of SEQ ID NO: 30, a complex (ribonucleoprotein) of a guide RNA specific to the target nucleotide sequence of the tomato-derived APX4 gene and an endonuclease protein;
  13. 제11항 또는 제12항의 방법에 의해 제조된 열매의 아스코르브산 함량이 증가된 유전체 교정 토마토 식물체.13. A genome-edited tomato plant with an increased content of ascorbic acid in the fruit produced by the method of claim 11 or 12.
  14. 제13항에 따른 토마토 식물체의 유전체가 교정된 종자.A seed in which the genome of the tomato plant according to claim 13 is corrected.
PCT/KR2021/019619 2020-12-23 2021-12-22 Solanum lycopersicum-derived apx4 gene to control ascorbic acid content of tomato fruits and use thereof WO2022139463A1 (en)

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