Tomatoes containing high levels of 7-dehydrocholesterol and method for their preparation

Abstract

The present invention relates to the tomato plant containing high levels of vitamin D3 precursor produced by using gene editing technology, and its preparation method.Specifically, it has been confirmed that the tomato plant that has deleted DWF5-1 gene accumulates high levels of 7-dehydrocholesterol and can produce seeds from it.Therefore, the tomato plant produced in this way contains high levels of vitamin D3 precursor and can have high commercial applicability.

Description

[Technical field]

[0001] The present invention relates to tomatoes containing high levels of 7-dehydrocholesterol (a precursor of vitamin D3) and methods for producing same. [Background technology]

[0002] Vitamin D is an essential nutrient for sustaining life. It has been reported that insufficient vitamin D increases the risk of not only rickets, osteomalacia, and osteoporosis, but also malignant tumors such as breast cancer, colorectal cancer, and prostate cancer; cardiovascular disease including hypertension; diabetes; multiple sclerosis; psoriasis; rheumatoid arthritis; and tuberculosis. Vitamin D is a vitamin that can be ingested through food but can also be synthesized in vivo through sufficient sunlight. Therefore, if there is insufficient exposure to sunlight, vitamin D-rich foods or vitamin D dietary supplements should be consumed.

[0003] In particular, modern people spend most of their day indoors, and even when they do outdoor activities, they block sunlight with clothing and hats, so they need to be careful about vitamin D deficiency. If exposure to sunlight is insufficient, they must get enough vitamin D through food, but there are not many foods with high vitamin D content. Even foods that are known to be rich in vitamin D contain only 0.29 μg per 100 g of about 7 slices of cheese (about 100 g), 2.9 μg per 100 g of tuna, 1.36 μg per 2 eggs (about 100 g), and 4.4 μg per 1 cup of raisins (about 100 g). Therefore, it is not possible to meet the daily recommended amount of vitamin D from these foods.

[0004] Furthermore, it has been reported that synthetic vitamin D as a vitamin D dietary supplement may have side effects and harmful effects, and natural vitamin D has the problem of being very expensive and difficult to obtain. Meanwhile, Korean Patent Application Publication No. 10-2017-0138657 discloses that when DWF5 (delta 5, 7-sterol deta 7 reductase, DWARF5), CPD (constitutive photomorphogenic DWAF, DWF3) and SMT1 (sterol methyltransferase 1) genes are deleted, lettuce rich in 7-dehydrocampesterol, a precursor of vitamin D, can be produced. However, no research has been conducted on the production of tomatoes rich in 7-dehydrocholesterol, a precursor of vitamin D3. Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, the present inventors conducted an experiment by deleting the DWF5-1, CPD and SMT1 genes by CRISPR technology. As a result, it was confirmed that when the DWF5-1 gene was deleted, tomatoes containing high concentrations of 7-dehydrocholesterol were produced. Based on the above, the present inventors have completed the present invention. Specifically, the present invention is to identify DWF5-1, CPD and SMT1 deletion sites optimized for developing tomatoes containing high concentrations of 7-dehydrocholesterol, which is a precursor for vitamin D3 production, and to develop tomatoes rich in vitamin D3 precursors containing them. [Means for solving the problem]

[0006] To achieve the above object, one aspect of the present invention provides a transformed tomato containing a high concentration of 7-dehydrocholesterol represented by the following formula 1:

[0007] In another aspect of the present invention, a vector is provided comprising one or more sgRNAs (single guide RNAs) that bind complementarily to a nucleotide sequence of the DWF5-1 gene and a nucleotide sequence encoding a CRISPR (clustered regularly interspaced palindromic repeats) associated protein.

[0008] In another aspect of the invention, there is provided a method for producing a transformed tomato containing elevated levels of 7-dehydrocholesterol, comprising introducing a vector into tomato using Agrobacterium. Effect of the Invention

[0009] When the transformed tomato produced by the present invention was homozygous for the DWF5-1 gene mutation, a large amount of 7-dehydrocholesterol was accumulated in the fruit and roots of the tomato. Furthermore, the homozygote (T2) was able to produce seeds, thus preserving the desired tomato traits. Therefore, the transformed tomato of the present invention has high commercial applicability. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 shows the sites in DWF5-1 targeted by DWF5-1 sgRNA. [Diagram 2] FIG. 1 shows the sites of the CPD targeted by the CPD sgRNA. [Diagram 3] FIG. 1 shows the sites of SMT1 targeted by SMT1 sgRNA. [Figure 4] FIG. 1 shows the results obtained by confirming the gene editing efficiency of DWF5-1 sgRNA using RGEN. [Diagram 5] FIG. 1 shows the results obtained by confirming the CPD gene editing efficiency of the CPD sgRNA using RGEN. [Figure 6] FIG. 1 shows the results obtained by confirming the SMT1 gene editing efficiency of SMT1 sgRNA using RGEN. [Figure 7] A schematic diagram of the Cas9-sgRNA vector loaded with DWF5-1 sgRNA is shown. [Figure 8] A schematic diagram of the Cas9-sgRNA vector loaded with DWF5-1 sgRNA and CPD sgRNA is shown. [Figure 9] A schematic diagram of the Cas9-sgRNA vector loaded with DWF5-1 sgRNA, CPD sgRNA and SMT1 sgRNA is shown. [Figure 10] 1 is a flow chart showing a process for constructing transformed tomatoes using Agrobacterium. [Figure 11] This shows the germination process of a tomato plant constructed through gene editing. [Figure 12] FIG. 1 shows results obtained by observing tomato plants edited with the DWF5-1 gene. [Figure 13] FIG. 1 shows the results obtained by observing tomato plants edited with the DWF5-1 and CPD genes. [Figure 14] FIG. 1 shows the results obtained by observing tomato plants edited with the DWF5-1, CPD and SMT1 genes. [Figure 15] FIG. 1 shows the results obtained by using the T7E1 assay to confirm whether the DWF5-1, CPD or SMT1 genes were edited. [Figure 16] FIG. 1 shows the results obtained by using Sanger sequencing to confirm whether the DWF5-1 gene was deleted. [Figure 17] FIG. 1 shows the results obtained by confirming the DWF5-1 gene phenotype in T1 generation transformed tomato plants in which the DWF5-1 gene has been deleted, by next-generation sequencing. [Figure 18a] FIG. 1 shows gene editing efficiency and amount of seeds harvested (top) and nucleotide sequence (bottom) of T1 generation homozygous transformed tomato plants with deleted DWF5-1 gene. [Figure 18b] FIG. 1 shows the nucleotide sequence of T1 generation homozygous transformed tomato plants with deleted DWF5-1 gene. [Figure 19] FIG. 1 is a flow chart showing the sample preparation process for measuring 7-dehydrocholesterol in T2 generation transformed tomato plants homozygous for the deletion of the DWF5-1 gene (T1). [Figure 20] FIG. 1 shows the measurement conditions by LC / MS for measuring 7-dehydrocholesterol contained in T2 generation transformed tomato plants of homozygotes (T1) lacking the DWF5-1 gene. [Figure 21] FIG. 1 shows the results obtained by measuring, by LC / MS, commercially available 7-dehydrocholesterol as a standard reagent, 7-dehydrocholesterol in wild-type (WT) tomato plants, and 7-dehydrocholesterol in transformed tomato plants of the T2 generation homozygous for the deletion of the DWF5-1 gene (T1). [Figure 22] FIG. 1 is a graph showing the results obtained by measuring 7-dehydrocholesterol in fruits of T2 generation transformed tomato plants homozygous for the deletion of the DWF5-1 gene (T1) by LC / MS. [Figure 23] FIG. 1 shows the results obtained by confirming, by RT-PCR, the expression of hygromycin and phosphinothricin (PPT) resistance genes in T2 generation transformed tomato plants homozygous for the deletion of the DWF5-1 gene (T1). [Figure 24]FIG. 1 shows the results obtained by analyzing the nucleotide sequence of the exon 6 domain of DWF5-1 in transformed tomato plants of the T2 generation (D100_3-14-29 and D100_7-1-15) that do not express an antibiotic resistance gene, in comparison with that of wild-type (WT) tomato plants. [Diagram 25] FIG. 1 shows the results obtained by analyzing the amino acid sequences of the exon 6 domain of DWF5-1 in transformed tomato plants of the T2 generation (D100_3-14-29 and D100_7-1-15) that do not express an antibiotic resistance gene, in comparison with that of wild-type (WT) tomato plants. [Figure 26] FIG. 1 shows the phenotype of homozygous (T2) transformed tomato plants (T2) lacking the DWF5-1 gene. [Figure 27] Graph showing the results obtained by confirming the expression levels of DWF5-1 or DWF5-2 genes in the roots, stems, leaves and fruits of tomato plants by q-PCR. ****p<0.0001 [Figure 28] FIG. 1 is a graph showing the results obtained by measuring 7-dehydrocholesterol in roots (cultured roots) of T3 generation transformed tomato plants homozygous for the deletion of the DWF5-1 gene (T1) by GC / MS. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Best Mode for Carrying Out the Invention In one aspect of the present invention, a transformed tomato is provided that contains high levels of 7-dehydrocholesterol of formula 1 below. [ka]

[0012] As used herein, the term "tomato" refers to a plant or its fruit having the scientific name Solanum lycopersicum and belonging to the family Solanaceae, order Solanales. Native to Central and South America, it is an annual plant with a height of 1-3 m and yellow flowers. The fruit is red due to lycopene and is used for edible purposes. As used herein, tomato may be a plant body, a plant organ (e.g., root, stem, leaf, petal (flower), seed, fruit, etc.), a plant tissue (epidermis, phloem, parenchyma, xylem, vascular tissue), etc. Specifically, it may be a fruit and / or a root. In this case, the tomato plant body may include a cell culture, including a root culture, etc.

[0013] As used herein, the term "7-dehydrocholesterol" is provitamin D3 that is converted to vitamin D3. In the present invention, the transformed tomato may contain 0.01 mg to 1.5 mg of 7-dehydrocholesterol per 100 g of weight. Specifically, the transformed tomato may contain 0.01 mg, 0.10 mg, 0.20 mg, 0.30 mg, 0.40 mg, 0.50 mg, 0.60 mg, 0.70 mg, 0.80 mg, 0.90 mg, 1.00 mg, 1.10 mg, 1.20 mg, 1.30 mg, 1.40 mg, or 1.50 mg of 7-dehydrocholesterol per 100 g of weight.

[0014] In the present invention, the transformed tomato may be genetically engineered to reduce the expression or activity of the Lycopersicum DWF5-1 (DWF5-1) gene or DWF5-1 protein compared to wild-type tomato, where the genetic engineering may be induced by modification of the nucleic acid sequence.

[0015] As used herein, the term "DWF5" refers to 7-dehydrocholesterol reductase. This enzyme is also called delta 5,7-sterol delta 7 reductase. This enzyme has the activity of reducing 5-dehydroepisterol, an intermediate metabolite of the phytosterol metabolic pathway, and converting it to 24-methylenecholesterol.

[0016] In Arabidopsis thaliana, where DWF5 exists as a single gene in plants, it is known that deletion of the DWF5 gene leads to a rapid decline in phytosterol metabolism, resulting in dwarfism that prevents growth to adulthood. In tomato, DWF5 exists in two forms, DWF5-1 and DWF5-2. In this case, DWF5-1 and DWF5-2 may contain the amino acid sequences of SEQ ID NO: 2 and SEQ ID NO: 4, respectively. It has been reported that the DWF5-1 gene functions in the phytosterol metabolic pathway, and the DWF5-2 gene functions in the cholesterol production pathway by reducing 7-dehydrocholesterol (7-dehydrocholesterol reductase) and converting it to cholesterol. For this reason, it can be expected that abnormal growth may occur when the DWF5-1 gene is deleted, which is likely to result in a decline in phytosterol metabolism. However, since it has been reported that the cholesterol pathway in plants evolved from the phytosterol metabolic pathway, the possibility that DWF5-1 and DWF5-2 have functional homology with each other cannot be completely excluded. As a result of the comparison and analysis of the gene expression patterns of DWF5-1 and DWF5-2 in the present invention, DWF5-1 was expressed 5 to 10 times higher than the DWF5-2 gene in all of the leaves, stems, roots, flowers, unripe fruits and mature fruits of tomato, and the DWF5-2 gene was also expressed at a low expression level in all tissues. Therefore, assuming the functional homology of DWF5-1 and DWF5-2, it was thought that the DWF5-1 gene could increase the 7-dehydrocholesterol content in tomato fruits more efficiently.

[0017] In the present invention, mutant plants in which only the DWF5-1 gene was deleted produced high amounts of provitamin D3 in both unripe and mature tomato fruits and root tissues, and their growth and seed production were similar to those of wild-type tomatoes (Figures 18, 26, and Table 13). Thus, in the present invention, a tomato that produces high concentrations of provitamin D3 in mature fruits due to deletion of at least the DWF5-1 gene was developed, and it was confirmed that single deletion of the DWF5-1 gene is an industrially useful technique that enables growth and fruit production similar to that of the wild type.

[0018] In the present invention, DWF5 can be DWF5-1. Specifically, in the present invention, DWF5 can be DWF5-1 comprising the amino acid sequence of SEQ ID NO: 2. DWF5-1 gene can be genomic DNA, cDNA or RNA comprising a nucleotide sequence encoding DWF5-1. More specifically, in the present invention, DWF5-1 gene can be genomic DNA. In the present invention, DWF5, DWF5-1 and SlDWF5-1 can be used interchangeably.

[0019] As used herein, the term "genomic DNA" refers to chromosomal DNA, the form in which genetic information is encoded in eukaryotic cells. Eukaryotic genomic DNA (hereinafter referred to as DNA) contains exons and introns. Exons are parts that contain nucleic acid sequences that code for proteins, and introns are parts that are not involved in protein synthesis. To produce various proteins required for the function of an organism, DNA is transcribed into RNA, in which exons, excluding introns, are linked together. Introns contain information that aids transcription, such as promoters that induce the start of transcription, and are used in the transcription process to create pre-mRNA, but are cut off rather than used to create mature mRNA.

[0020] In the present invention, the DWF5-1 gene may comprise 12 exons and 11 introns. Specifically, DWF5-1 may comprise exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, or exon 12. In addition, DWF5-1 may comprise intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, intron 7, intron 8, intron 9, intron 10, or intron 11.

[0021] More specifically, exon 1 to exon 12 of the DWF5-1 gene may comprise or consist of the nucleic acid sequences of SEQ ID NO: 40 to SEQ ID NO: 51, respectively. Intron 1 to intron 11 of the DWF5-1 gene may comprise or consist of the nucleic acid sequences of SEQ ID NO: 52 to SEQ ID NO: 62, respectively.

[0022] In one embodiment, in the present invention, the DWF5-1 gene may comprise or consist of the nucleic acid sequence of SEQ ID NO:63.

[0023] In the present invention, the DWF5-1 gene may contain a flanking region that regulates the transcription of DWF5-1.

[0024] As used herein, the term "flanking region" refers to a DNA sequence that flanks both sides of a particular gene and is not transcribed into RNA. The DNA region adjacent to the 5' end of a gene is referred to as the 5' flanking region, and the DNA region adjacent to the 3' end of a gene is referred to as the 3' flanking region. Flanking regions contain regulatory sequences and regulate the transcription of genes by binding to proteins involved in transcription via the above sequences. In particular, in eukaryotes, 5' flanking regions contain enhancers, silencers, promoters, etc., and regulate transcription by binding to proteins such as transcription factors and RNA polymerase.

[0025] In one embodiment of the present invention, the 5' flanking region of the DWF5-1 gene may comprise the nucleic acid sequence of SEQ ID NO: 64. In one embodiment, the 3' flanking region of the DWF5-1 gene may comprise the nucleic acid sequence of SEQ ID NO: 65.

[0026] More specifically, in the present invention, the DWF5-1 gene may be a cDNA. In one embodiment, it may comprise or consist of the nucleic acid sequence of SEQ ID NO:1.

[0027] As used herein, the term "genetic engineering" or "genetically engineered" refers to the act of introducing one or more genetic modifications into a cell or into a cell produced thereby.

[0028] As used herein, the term "reduced expression or activity of a gene or protein" means that the expression or activity of a target gene or protein is lower than the expression or activity of the wild-type of the same species to which the target gene or protein corresponds. Furthermore, "inactivated" means that a protein is produced that is not expressed or that is expressed but has no activity compared to the wild-type target gene or protein.

[0029] In other words, the reduction or inactivation of the expression or activity of the DWF5-1 gene or protein means that all or part of the biological functions or roles normally performed by the DWF5-1 gene in wild-type tomato are lost. For example, the protein expressed by the DWF5-1 gene may be terminated prematurely or lose its normal function as a protein. Specifically, gene editing may be to knock out the DWF5-1 gene by generating a stop codon in the target region or by generating a codon that codes for an amino acid different from that of the wild type. In addition, it may be, but is not limited to, to introduce a mutation into a non-coding DNA sequence that does not produce a protein.

[0030] Specifically, the genetically engineered transformed tomato may have a DWF5-1 gene or protein that is reduced by about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, or about 100% compared to the DWF5-1 gene or protein of a wild-type tomato. Expression or activity of the DWF5-1 gene or DWF5-1 protein may be determined using any method known in the art.

[0031] The genetic engineering may be induced by modification of the nucleic acid sequence. Specifically, the genetic engineering may be induced by modification of the nucleic acid sequence by substitution (conversion), deletion or insertion. Through the genetic engineering, the DWF5-1 gene of the transformed tomato of the present invention may contain a gene sequence different from that of the wild-type tomato DWF5-1 gene, and may lose the biological function of the gene.

[0032] More specifically, the manipulation of the DWF5-1 gene can be induced by any one modification in the nucleic acid sequence selected from the group consisting of: i) deletion of all or 1 to 50 consecutive nucleotide sequences of the DWF5-1 gene; ii) replacing nucleotides 1 to 50 of the DWF5-1 gene with nucleotides different from those of the wild-type DWF5-1 gene; iii) an insertion of 1 to 50 nucleotides, each independently selected from A, T, C, and G, into the DWF5-1 gene; and iv) combinations thereof.

[0033] More specifically, the genetic engineering can be induced by any one modification in a nucleic acid sequence selected from the group consisting of: i) a deletion of all or part of the DWF5-1 gene, for example a deletion of one or more nucleotides of the DWF5-1 gene, for example a deletion of 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-3 or 1 nucleotide; ii) replacement of one or more nucleotides, e.g., 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-3 or 1 nucleotide, of the DWF5-1 gene with a nucleotide different from that of the wild-type DWF5-1 gene; iii) insertion of one or more nucleotides selected from A, T, C and G, for example, 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 3 or 1 nucleotide, into any site of the DWF5-1 gene; and iv) A combination of i) to iii).

[0034] The portion of the nucleic acid sequence of the DWF5-1 gene (the "target region") to be modified can be a region of 1 or more, 3 or more, 5 or more, 7 or more, 10 or more, 12 or more, 15 or more, 17 or more, 20 or more, 25 or more, 27 or more, 30 or more, 33 or more, 37 or more, 40 or more, 43 or more, 47 or more, or 50 or more consecutive nucleotides in the gene.

[0035] In one embodiment of the present invention, the modification in the nucleic acid sequence of the DWF5-1 gene can be included in the nucleic acid sequence of any one of the domains selected from the group consisting of exon 6 (SEQ ID NO: 9), exon 7 (SEQ ID NO: 10), exon 9 (SEQ ID NO: 11), exon 10 (SEQ ID NO: 12), exon 11 (SEQ ID NO: 13) and combinations thereof. In one embodiment of the present invention, the modification in the nucleic acid sequence of the DWF5-1 gene can be included in the nucleic acid sequence of the exon 6 domain. Specifically, the nucleotide sequence of SEQ ID NO: 9, which includes the nucleic acid sequence of the exon 6 domain of DWF5-1, can be modified by substitution, deletion or insertion in the nucleic acid sequence. More specifically, the modification in the sequence can be induced by substitution, deletion or insertion in the nucleotide sequence of SEQ ID NO: 9, which includes the nucleic acid sequence of the exon 6 domain of DWF5-1 gene, to reduce the expression or activity of the DWF5-1 gene or DWF5-1 protein, or to cause the presence of a stop codon.

[0036] The transformed tomato of the present invention may be further genetically modified to reduce the expression or activity of the CPD gene or CPD protein. The genetic modification is the same as described above.

[0037] As used herein, the term "CPD (Constitutive Photomorphogenic DWAF)" refers to an enzyme of the CP90A family of cytochrome P450 monooxygenases, also known as DWF3. During the brassinolide biosynthesis process, it acts on the C6 oxidation pathway to convert cathasterone to teasterone and 6-deoxycathasterone to 6-deoxyteasterone. In the present invention, the enzyme serves to reduce 7-dehydrocampesterol to 7-dehydrocampestanol.

[0038] In this case, the CPD may comprise the amino acid sequence of SEQ ID NO: 6. Additionally, the nucleic acid encoding it may comprise the nucleotide sequence of SEQ ID NO:5.

[0039] In the present invention, the reduction or inactivation of the expression or activity of the CPD gene or CPD protein means that all or part of the biological functions or roles normally performed by the CPD gene in wild-type tomato are lost. In this case, the reduction and inactivation of the expression or activity of the gene or protein are the same as described above. For example, the protein expressed by the CPD gene may be prematurely terminated or lose its normal function as a protein. Specifically, gene editing may be to knock out the CPD gene by generating a stop codon in the target region or by generating a codon that codes for an amino acid different from that of the wild type. In addition, it may be, but is not limited to, to introduce a mutation into a non-coding DNA sequence that does not produce a protein.

[0040] Specifically, the genetically engineered transformed tomato may have a CPD gene or protein that is reduced by about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, or about 100% compared to the CPD gene or protein of a wild-type tomato. Expression or activity of a CPD gene or CPD protein may be determined using any method known in the art.

[0041] Genetic engineering may be induced by modification of the nucleic acid sequence. Specifically, genetic engineering may be induced by modification of the nucleic acid sequence by substitution, deletion or insertion. Through the genetic engineering, the CPD gene of the transformed tomato of the present invention may contain a gene sequence different from that of the wild-type tomato CPD gene, and may lose the biological function of the gene.

[0042] More specifically, the manipulation of the CPD gene can be induced by any one modification in the nucleic acid sequence selected from the group consisting of: i) deletion of all or 1 to 50 consecutive nucleotide sequences of the CPD gene; ii) replacement of nucleotides 1 to 50 of the CPD gene with nucleotides different from those of the wild-type CPD gene; iii) insertion of 1 to 50 nucleotides, each independently selected from A, T, C, and G, into the CPD gene; and iv) combinations thereof.

[0043] More specifically, the genetic engineering can be induced by any one modification in a nucleic acid sequence selected from the group consisting of: i) a deletion of all or part of the CPD gene, for example a deletion of one or more nucleotides of the CPD gene, for example a deletion of 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-3 or 1 nucleotide; ii) replacement of one or more nucleotides of the CPD gene, e.g. 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-3 or 1 nucleotide, with a nucleotide different from that of the wild-type CPD gene; iii) insertion of one or more nucleotides selected from A, T, C and G, for example, 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 3 or 1 nucleotide, into any site of the CPD gene; and iv) A combination of i) to iii).

[0044] The portion of the nucleic acid sequence of the CDP gene (the "target region") to be modified can be a region of 1 or more, 3 or more, 5 or more, 7 or more, 10 or more, 12 or more, 15 or more, 17 or more, 20 or more, 25 or more, 27 or more, 30 or more, 33 or more, 37 or more, 40 or more, 43 or more, 47 or more, or 50 or more contiguous nucleotides in the gene.

[0045] In one embodiment of the present invention, the modification in the nucleic acid sequence of the CPD gene may be included in the nucleic acid sequence of any one of the domains selected from the group consisting of exon 7 (SEQ ID NO: 14), exon 8 (SEQ ID NO: 15) and combinations thereof. In one embodiment of the present invention, the modification in the nucleic acid sequence of the CPD gene may be a modification in the nucleic acid sequence of the exon 8 domain. Specifically, the nucleotide sequence of SEQ ID NO: 15, which includes the nucleic acid sequence of the exon 8 domain of the CPD gene, may be modified by substitution, deletion or insertion in the nucleic acid sequence. More specifically, the modification in the sequence may be induced by substitution, deletion or insertion in the nucleotide sequence of SEQ ID NO: 15, which includes the nucleic acid sequence of the exon 8 domain of the CDP gene, to reduce the activity of CDP or to cause the presence of a stop codon.

[0046] The transformed tomato containing mutations in the DWF5-1 and CPD genes of the present invention may be further genetically engineered to reduce the expression or activity of the SMT1 gene or SMT1 protein. The genetic engineering is the same as described above.

[0047] As used herein, the term "SMT1 (sterol methyltransferase 1)" refers to an enzyme that adds a methyl group to the side branch of a steroid. Specifically, SMT1 serves to convert 5-dehydroepisterol to 7-dehydrocampesterol.

[0048] In this case, SMT1 may comprise the amino acid sequence of SEQ ID NO: 8. Additionally, the nucleic acid encoding it may comprise the nucleotide sequence of SEQ ID NO: 7.

[0049] In the present invention, the reduction or inactivation of the expression or activity of the SMT1 gene or SMT1 protein means that all or part of the biological functions or roles normally performed by the SMT1 gene in wild-type tomato are lost. In this case, the reduction and inactivation of the expression or activity of the gene or protein are the same as above. For example, the protein expressed by the SMT1 gene may be terminated prematurely or lose its normal function as a protein. Specifically, gene editing may be to knock out the SMT1 gene by generating a stop codon in the target region or by generating a codon that codes for an amino acid different from that of the wild type. In addition, it may be, but is not limited to, to introduce a mutation into a non-coding DNA sequence that does not produce a protein.

[0050] Specifically, the genetically engineered transformed tomato may have an SMT1 gene or protein that is reduced by about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, or about 100% compared to the SMT1 gene or SMT1 protein of a wild-type tomato. Expression or activity of the SMT1 gene or SMT1 protein may be determined using any method known in the art.

[0051] Genetic engineering may be induced by modification of nucleic acid sequence. Specifically, genetic engineering may be induced by modification of nucleic acid sequence by substitution, deletion or insertion. Through said genetic engineering, the SMT1 gene of the transformed tomato of the present invention may contain a gene sequence different from that of the wild-type tomato SMT1 gene, and may lose the biological function of said gene.

[0052] More specifically, the manipulation of the SMT1 gene can be induced by any one modification in the nucleic acid sequence selected from the group consisting of: i) deletion of all or a 1-50 contiguous nucleotide sequence region of the SMT1 gene; ii) replacement of nucleotides 1 to 50 of the SMT1 gene with nucleotides different from those of the wild-type SMT1 gene; iii) an insertion into the SMT1 gene of 1 to 50 nucleotides, each independently selected from A, T, C, and G; and iv) combinations thereof.

[0053] More specifically, the genetic engineering can be induced by any one modification in a nucleic acid sequence selected from the group consisting of: i) a deletion of all or part of the SMT1 gene, for example a deletion of one or more nucleotides of the SMT1 gene, for example a deletion of 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-3 or 1 nucleotide; ii) replacement of one or more nucleotides of the SMT1 gene, e.g., 1-50, 1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 1-5, 1-3 or 1 nucleotide, with a nucleotide different from that of the wild-type SMT1 gene; iii) insertion of one or more nucleotides selected from A, T, C and G, for example, 1 to 50, 1 to 45, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 3 or 1 nucleotide, into any site of the SMT1 gene; and iv) A combination of i) to iii).

[0054] The portion of the nucleic acid sequence of the SMT1 gene (the "target region") to be modified can be a region of 1 or more, 3 or more, 5 or more, 7 or more, 10 or more, 12 or more, 15 or more, 17 or more, 20 or more, 25 or more, 27 or more, 30 or more, 33 or more, 37 or more, 40 or more, 43 or more, 47 or more, or 50 or more contiguous nucleotides in the gene.

[0055] In one embodiment of the present invention, the modification in the nucleic acid sequence of the SMT1 gene may be included in the nucleic acid sequence of any one of the domains selected from the group consisting of exon 7 (SEQ ID NO: 16), exon 9 (SEQ ID NO: 17), exon 11 (SEQ ID NO: 18), exon 12 (SEQ ID NO: 19) and combinations thereof. In one embodiment of the present invention, the mutation in the SMT1 gene may include a modification in the nucleic acid sequence of the exon 7 and exon 9 domains. Specifically, the mutation in the SMT1 gene may be a modification by substitution, deletion or insertion in the nucleotide sequence of SEQ ID NO: 16 and SEQ ID NO: 17, which includes the nucleic acid sequence of the exon 7 and exon 9 domains. More specifically, the modification of the sequence may be induced by substitution, deletion or insertion in the nucleotide sequence of SEQ ID NO: 16 and SEQ ID NO: 17, which includes the nucleic acid sequence of the exon 7 and exon 9 domains of the SMT1 gene, to reduce the activity of SMT1 or to result in the presence of a stop codon.

[0056] The transformed tomato may be homozygous. That is, the modification of the nucleic acid sequence may be included in two genes encoding DWF5-1. In one embodiment, the modification of the nucleic acid sequence may be included in two genes encoding any one domain selected from the group consisting of exon 6, exon 7, exon 9, exon 10, exon 11, and combinations thereof of the DWF5-1 gene. In one embodiment of the present invention, the transformed tomato may have a modified sequence in two genes encoding the exon 6 domain of the DWF5-1 gene that is different from the nucleic acid sequence of the wild-type DWF5-1 gene.

[0057] In the transformed tomato, the modification of the nucleic acid sequence may be included in two genes encoding DWF5-1, CPD and SMT1. In this case, the transformed tomato may have a modified sequence in the two genes encoding any one domain selected from the group consisting of exon 7, exon 8 and combinations thereof of the CPD gene, which is different from the nucleic acid sequence of the wild-type CPD gene. Furthermore, the modification of the nucleic acid sequence may be included in the two genes encoding any one domain selected from the group consisting of exon 7, exon 9, exon 11, exon 12 and combinations thereof of the SMT1 gene. In this case, the modification of the nucleic acid sequence is the same as above.

[0058] In one embodiment of the present invention, in a transformed tomato, nucleic acid sequence modifications may be included in two genes encoding the exon 6 domain of DWF5-1, the exon 7 domain of CDP, and the exon 7 and exon 9 domains of SMT1, respectively.

[0059] When the transformed tomato of the present invention is homozygous and the expression or activity of the DWF5-1 gene or protein is reduced or inactivated, the tomato can produce seeds. When the transformed tomato is homozygous and the expression or activity of the DWF5-1, CPD and SMT1 genes or proteins is reduced or inactivated, the tomato may not produce fruit and may not produce seeds.

[0060] In another aspect of the present invention, a vector is provided, which comprises the nucleotide sequence of DWF5-1 gene and one or more sgRNAs that are complementary to the nucleotide sequence of coding CRISPR-associated protein.Wherein, DWF5-1 is the same as above.Furthermore, DWF5-1 gene can be genomic DNA, cDNA or RNA.

[0061] As used herein, the term "guide RNA (gRNA)" refers to a polynucleotide that recognizes a target nucleic acid in a cell and cleaves, inserts, or ligates the target nucleic acid through genome editing. The gRNA may include a sequence complementary to a target sequence in the target nucleic acid. The gRNA may be a polynucleotide complementary to a nucleotide sequence of 2 to 24 consecutive nucleotides (hereinafter referred to as "nt") in the 5' or 3' direction of the PAM in the target nucleic acid. The length of the gRNA may be 17 nt, 18 nt, 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, or 24 nt.

[0062] The gRNA may be a single guide RNA (sgRNA). The sgRNA may include a CRISPR RNA (crRNA) specific to a target nucleic acid sequence and / or a transactivating crRNA (tracrRNA) that forms a complex with a CRISPR-associated protein. The sgRNA may include a portion having a nucleotide sequence (targeting sequence) complementary to a target nucleic acid sequence (targeting region) in a target gene (also referred to as a spacer region, a target DNA recognition sequence, a base pairing region, etc.) and a hairpin structure for binding to a CRISPR-associated protein. More specifically, the sgRNA may include a portion including a nucleotide sequence (targeting sequence) complementary to a target nucleic acid sequence in a target gene, a hairpin structure for binding to a CRISPR-associated protein, and a terminator sequence. The above structures may be present sequentially from 5' to 3', but are not limited thereto. Any type of sgRNA may be used as long as the sgRNA includes the main regions of the crRNA and tracrRNA and a nucleotide sequence complementary to the target gene.

[0063] In the present invention, the sgRNA can be complementary to the nucleic acid sequence of the genomic DNA of DWF5-1. Specifically, the sgRNA can be complementary to SEQ ID NO: 63. More specifically, the sgRNA can be complementary to the nucleic acid sequence of one or more domains selected from the group consisting of exon 1 to exon 12, intron 1 to intron 11, 5' flanking region and 3' flanking region of the DWF5-1 gene. More specifically, it can be complementary to the nucleic acid sequence of one or more domains selected from the group consisting of SEQ ID NO: 40 to SEQ ID NO: 62, SEQ ID NO: 64 and SEQ ID NO: 65.

[0064] In the present invention, the sgRNA can be complementary to the nucleic acid sequence of the cDNA of DWF5-1. Specifically, the sgRNA can be complementary to the nucleic acid sequence of SEQ ID NO:1.

[0065] In one embodiment of the present invention, the sgRNA may be complementary to the nucleic acid sequence of any one of the domains selected from the group consisting of exon 6, exon 7, exon 9, exon 10 and exon 11 of the DWF5-1 gene. In one embodiment, the sgRNA binding to the nucleic acid sequence of the exon 6 domain of the DWF5-1 gene may comprise the nucleotide sequence of SEQ ID NO: 20 or SEQ ID NO: 21. In one embodiment, the sgRNA binding to the nucleic acid sequence of the exon 7 domain may comprise the nucleotide sequence of SEQ ID NO: 22. In one embodiment, the sgRNA binding to the nucleic acid sequence of the exon 9 domain may comprise the nucleotide sequence of SEQ ID NO: 23 or SEQ ID NO: 24. In one embodiment, the sgRNA binding to the exon 10 domain may comprise the nucleotide sequence of SEQ ID NO: 25. In one embodiment, the sgRNA binding to the nucleic acid sequence of the exon 11 domain may comprise the nucleotide sequence of SEQ ID NO: 26. Preferably, in one embodiment of the present invention, the sgRNA may comprise or consist of the nucleotide sequences of SEQ ID NO: 20 and SEQ ID NO: 21, which are complementary to the nucleic acid sequence of the exon 6 domain.

[0066] In the present invention, the vector may further comprise an sgRNA that binds complementarily to the nucleotide sequence of the CPD gene. The sgRNA that binds complementarily to the CPD gene may bind complementarily to the nucleotide sequence of the exon 7 or exon 8 domain of the CPD gene. The sgRNA that binds to the exon 7 domain of the CPD gene may comprise the nucleotide sequence of SEQ ID NO: 27. The sgRNA that binds to the exon 8 domain of the CPD gene may comprise the nucleotide sequence of SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 or SEQ ID NO: 31. In one embodiment of the present invention, the sgRNA may comprise the nucleotide sequence of SEQ ID NO: 28 and SEQ ID NO: 29 that bind complementarily to the nucleotide sequence of the exon 8 domain.

[0067] In the present invention, the vector comprising the sgRNA that binds complementarily to the nucleotide sequence of the DWF5 and CPD genes may further comprise an sgRNA that binds complementarily to the nucleotide sequence of the SMT1 gene. In this case, the sgRNA that binds complementarily to the nucleotide sequence of the SMT1 gene may bind complementarily to the nucleotide sequence of one or more domains selected from the group consisting of exon 7, exon 9, exon 11 and exon 12 of the SMT1 gene. The sgRNA that binds to the exon 7 domain of the SMT1 gene may comprise the nucleotide sequence of SEQ ID NO: 32. The sgRNA that binds to the exon 9 domain of the SMT1 gene may comprise the nucleotide sequence of SEQ ID NO: 33. The sgRNA that binds to the exon 11 domain of the SMT1 gene may comprise the nucleotide sequence of SEQ ID NO: 34. The sgRNA that binds to the exon 12 domain of the SMT1 gene may comprise the nucleotide sequence of SEQ ID NO: 35. In one embodiment of the present invention, the sgRNA may comprise the nucleotide sequences of SEQ ID NO: 32 and SEQ ID NO: 33 that bind complementarily to the nucleotide sequences of the exon 7 and exon 9 domains. In this case, the sgRNAs that bind complementary to the nucleotide sequences of the DWF5 and CPD genes are the same as described above.

[0068] As used herein, the term "CRISPR (clustered regularly interspaced palindromic repeats) associated protein" (CRISPR-associated protein) refers to an enzyme that can recognize and cleave nucleic acids, such as DNA or RNA, when the nucleic acid has double strands or single strands (dsDNA / RNA and ssDNA / RNA). Specifically, it can recognize and cleave double-stranded or single-stranded nucleic acids bound to sgRNA. CRISPR-associated proteins include CRISPR-associated proteins and variants thereof that have their functions.

[0069] CRISPR-related proteins are found in Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Nocardiopsis dassonvillei, Streptomyces pristinae spiralis, Streptomyces viridochromogenes, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginosa, Pseudomonas aeruginosa, Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus It may be derived from the bacteria chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, Acaryochloris marina, Leptotrichia shahii, Prevotella or Francisella novicida. .

[0070] In the present invention, the CRISPR-associated proteins include Cas9, Cpf1, c2c1, C2c2, Cas13, c2c3, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a, Cas8a1, Cas8a2, Cas8b, Cas8c, Csn1, Csx12, Cas10, Cas10d, Cas10, Cas10d, CasF, CasG, CasH, Csyl, Csy2, Csy3, Cse1 (C asA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csxl, Csx15, Csf1, Csf2, Csf3, Csf4 and Cul966. Specifically, the CRISPR-associated protein may be Cas9.

[0071] The Cas9 protein can form a complex with the guide sgRNA and act as a form of ribonucleoprotein (RNP), where "ribonucleoprotein (RNP)" is a complex of RNA and protein, which in the present invention can be the Cas9-sgRNA complex.

[0072] As used herein, the term "vector" refers to a nucleic acid vehicle that can be introduced into a host cell and then recombined and inserted into the genome of the host cell. Alternatively, vector is understood to be a nucleic acid vehicle that contains a polynucleotide sequence that can autonomously replicate as an episome. Such vectors include linear nucleic acid, plasmid, phagemid, cosmid, RNA vector, viral vector, minichromosome and their analogs. Examples of viral vectors include, but are not limited to, retrovirus, adenovirus and adeno-associated virus.

[0073] In one embodiment of the present invention, the vector may comprise any one of the nucleotide sequences of SEQ ID NO: 36 to SEQ ID NO: 38. Furthermore, in the present invention, the vector has about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% sequence homology with any one of SEQ ID NO: 36 to SEQ ID NO: 38. Furthermore, the vector may be partially or completely codon-optimized for expression in a target organism or cell.

[0074] The vector may be operably linked to a suitable promoter so that the polynucleotide can be expressed in the host cell. When the vector of the present invention is applied to a plant cell, the promoter of the present invention may include a promoter used for gene introduction into the plant body. Examples of promoters may include, but are not limited to, SP6 promoter, T7 promoter, T3 promoter, PM promoter, corn ubiquitin promoter (Ubi), cauliflower mosaic virus (CaMV) 35S promoter, nopaline synthase (nos) promoter, Scrophulariaceae mosaic virus 35S promoter, sugarcane basiliform virus promoter, Comelina yellow mottle virus promoter, ribulose-1,5-bisphosphate carboxylase small subunit (ssRUBISCO) light-inducible promoter, rice cytosolic triosephosphate isomerase (TPI) promoter, Arabidopsis adenine phosphoribosyltransferase (APRT) promoter, and octopine synthase promoter.

[0075] Specifically, the vector may be a plasmid DNA, a phage DNA, etc. Furthermore, the vector may be a commercially developed plasmid (pUC18, pBAD, pIDTSAMRT-AMP, etc.), an Escherichia coli-derived plasmid (pYG601BR322, pBR325, pUC118, pUC119, etc.), a Bacillus subtilis-derived plasmid (pUB110, pTP5, etc.), a yeast-derived plasmid (YEp13, YEp24, YCp50, etc.), a phage DNA (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.), an animal virus vector (retrovirus, adenovirus, vaccinia virus, etc.), an insect virus vector (baculovirus, etc.), etc. The expression level and modification of the protein of the vector vary depending on the host cell, so it is preferable to select and use the host cell most suitable for the purpose.

[0076] In the present invention, the vector may contain a selection marker. A selection marker is a nucleic acid sequence having a characteristic that can be selected by conventional chemical methods, and includes all genes that can distinguish transformed cells from non-transformed cells. Examples of markers may include, but are not limited to, herbicide resistance genes such as glyphosate, glufosinate ammonium or phosphinothricin, and antibiotic resistance genes such as kanamycin, G418, bleomycin, hygromycin and chloramphenicol. In one embodiment of the present invention, the selection marker may be hygromycin and / or phosphinothricin.

[0077] In another aspect of the present invention, a method is provided for producing a transformed tomato containing high concentrations of 7-dehydrocholesterol, comprising introducing a vector containing a gene encoding an sgRNA and a gene encoding a CRISPR-associated protein into tomato using Agrobacterium.

[0078] The transformed plant may be constructed by a plant transformation method known in the art. Those skilled in the art can select and implement a known transformation method suitable for a particular plant, taking into consideration the characteristics of the plant selected as the host. Specifically, a transformation method using Agrobacterium may be used as a plant transformation method.

[0079] "Transformation method using Agrobacterium" refers to a method of transforming a foreign gene into a plant cell using Agrobacterium, a gram-negative soil bacterium that causes tumors in the roots and stems of plants. This method uses the phenomenon of inserting the T-DNA (transfer DNA) of the tumor-inducing plasmid (Ti plasmid) found in Agrobacterium such as Agrobacterium tumefaciens and Agrobacterium rhizogenes into the plant genome.

[0080] In one embodiment of the present invention, when the nucleic acid sequence modification is contained in the exon 6 domain of the DWF5-1 gene, homozygous tomatoes (T1) can produce seeds (Figure 18). Furthermore, homozygous T2 generation tomatoes with modifications in the nucleic acid sequence in the exon 6 domain of the DWF5-1 gene grow normally and produce seeds (Figure 26), and 7-dehydrocholesterol accumulates in fruits and roots at high levels (Table 14, Figures 21 and 22). However, when the transformed tomatoes contained modifications in the nucleic acid sequence in the exon 6 domain of the DWF5-1 gene and the exon 8 domain of the CPD gene, the tomatoes could not grow into intact plants. Furthermore, when the transformed tomatoes were homozygous with modifications in the nucleic acid sequence in the exon 6 domain of the DWF5-1 gene, the exon 8 domain of the CPD gene, the exons 7 and 9 domains of the SMT1 gene, no fruits were produced. EXAMPLES

[0081] The present invention will now be described in more detail with reference to the following examples. However, the following examples are only for the purpose of illustrating the present invention, and the scope of the present invention is not limited to these examples.

[0082] Example 1. Construction of Cas9-sgRNA vectors for gene editing of DWF5, CPD and SMT1 The design and selection of sgRNAs for gene editing of DWF5-1 (hereafter referred to as "DWF5", Figure 1), CPD (Figure 2) and SMT1 (Figure 3) were performed through the Cas-Designer website (http: / / www.rgenome.net / cas-dessdaigner / ). The name or sequence of the target gene and the animal or plant species were selected, and then the Cas9 to be used was selected, and the sgRNA was designed based on the required exon site in the gene, the GC content of the sgRNA, and the off-target consequences when mismatches are present. Cas9 has a PAM sequence of NGG-3', and tomato (S. lycopersicum) was selected as the plant (Figures 1-3). Furthermore, the sgRNAs were screened to confirm the gene editing efficiency (Table 2 and Figures 1-3).

[0083] Specifically, RGEN (RNA-guided endonuclease) analysis was used to verify the validity of sgRNA targeting. The DNA template was purified from the above-mentioned amplified PCR product (PCR product) using a PCR purification kit, and then the amount of DNA template was measured, and RGEN was performed in the combination shown in Table 1. After incubation at 37 ° C for 1 hour, 3 μL of 6 × DNA-Purple dye was added to each, frozen, and then loaded onto a 2.5% gel. Gene editing efficiency was analyzed by analyzing the degree of uncut bands in the DNA template.

[0084] [Table 1]

[0085] As a result, it was confirmed that in the case of sgRNAs constructed targeting the DWF5 gene, all sgRNAs except D5-4 and D5-6 were able to target the DWF5 gene (Figure 4). All CPD sgRNAs except C1 targeted the CPD gene (Figure 5), and all of the constructed SMT1 sgRNAs targeted the CPD gene (Figure 6).

[0086] [Table 2] TIFF2025503326000005.tif60170

[0087] Based on the results of the sgRNA screening, Cas9-sgRNA vectors were finally constructed containing sgRNAs designed to target DWF5, CPD or SMT1. The pNGPJ0014 vector (SEQ ID NO: 39) was constructed based on the pCAMBIA1300 plasmid carrying kanamycin resistance and hygromycin resistance genes.

[0088] The pNGPJ0014 vector has an antibiotic cassette for selection with kanamycin and hygromycin antibiotics, and a polycistronic tRNA-gRNA cassette synthesized using Cas9. Cas9 was constructed to be expressed by the Arabidopsis ubiquitin 10 (Ubi10) promoter, and the tRNA-gRNA was constructed to be expressed by the Arabidopsis ubiquitin 6 promoter.

[0089] To deliver the Cas9 protein to the nucleus, the SV40 (PKKKRKV) nuclear localization signal sequence was added to the N-terminus of the Cas9 open reading frame, and the Bipartite (KEPAATKKAGQAKKKK) nuclear localization signal sequence was added to the C-terminus. At this time, Cas9-sgRNA vectors targeting one to three types of genes were constructed. In addition, they were constructed to load two types of sgRNA per target. The Cas-sgRNA vector containing the nucleotide sequences of D5-1 sgRNA (SEQ ID NO: 20) and D5-2 sgRNA (SEQ ID NO: 21) targeting the DWF5 gene was named "D100" (SEQ ID NO: 36) (Figure 7).

[0090] The Cas9-sgRNA vector targeting the CPD and DWF5 genes was named "D120" (SEQ ID NO: 37), where the CPD sgRNA comprises the nucleotide sequences of C2 (SEQ ID NO: 28) and C3 (SEQ ID NO: 29), and the DWF5 sgRNA comprises the nucleotide sequences of D5-1 (SEQ ID NO: 20) and D5-2 (SEQ ID NO: 21) (Figure 8).

[0091] The Cas9-sgRNA vector targeting the SMT1, CPD and DWF5 genes was named "D121" (SEQ ID NO: 38), where the SMT1 sgRNA comprises the nucleotide sequences of S1 (SEQ ID NO: 32) and S2 (SEQ ID NO: 33), the CPD sgRNA comprises the nucleotide sequences of C2 (SEQ ID NO: 28) and C3 (SEQ ID NO: 29), and the DWF5 sgRNA comprises the nucleotide sequences of D5-1 (SEQ ID NO: 20) and D5-2 (SEQ ID NO: 21) (Figure 9).

[0092] Example 2. Transformation of tomato by gene editing of DWF5, CPD and SMT1 Tomato transformation was performed as shown in the schematic diagram of Figure 10. The media and hormones used at each step were prepared and used in the same manner as those described in Tables 3 to 8.

[0093] [Table 3]

[0094] [Table 4]

[0095] [Table 5]

[0096] [Table 6]

[0097] [Table 7]

[0098] [Table 8]

[0099] [Table 9]

[0100] Specifically, tomato seeds were disinfected with 70% ethanol for 5 seconds and disinfected with 2% bleach for 20 minutes. The disinfected seeds were washed 4-5 times with sterile water and then placed on sterile filter paper to remove moisture. The disinfected tomato seeds were transferred to the sowing medium TMS (Table 3) and cultured under dark culture conditions at 25 °C for 3 days, and then cultured under light culture conditions. After sowing, when the cotyledons grew to 1 cm-1.5 cm (9-12 days), the apex of the tomato hypocotyl was cut to 0.7 cm-1.0 cm, and about 18-20 pieces (4, 5, 5 and 4, or 5, 5, 5 and 5, a total of 18-20 pieces, respectively) were planted on a TPC medium plate (Table 4) and cultured under light culture conditions (16L / 8D) for 1 day.

[0101] Agrobacteria (GV3101 strain, DSMZ, catalog number DSM12364) was cultured in 50 mL of bacterial culture medium (YEP) containing 100 mM AS (acetosyringone), 50 mg / L rifampicin, and 50 mg / L kanamycin (Km) under dark culture conditions at 28°C for 1 to 2 days, until the OD600 value reached 0.8 to 1.2.

[0102] The cultured bacteria were centrifuged at 6,000 rpm for 15 min at 4°C, the supernatant was removed, and the pellet was resuspended and diluted in an equal volume of tomato co-culture (TCC) medium (Table 5) to give an OD value of 0.5.

[0103] 40 mL of the bacterial culture (OD600 value of 0.5) prepared by the above method and the plant bodies pretreated under light culture conditions were placed in a 50 mL tube (based on the sowing of 200 seeds) and cultured at 25 ° C and room temperature for 20 to 30 minutes. The co-cultured explants were transferred to sterile filter paper, dried to remove as much water as possible, and then about 18 to 20 pieces (4, 5, 5 and 4, or 5, 5, 5 and 5, totaling 18 to 20 pieces, respectively) were planted on a TCC medium plate (Table 5) and cultured at 25 ° C and under light culture conditions (16 L / 8 D) for 2 days. After 2 days of co-culture, the explants were transferred to a shoot selection medium (TSI, Table 7) and then cultured at 25 ° C and under light culture conditions (16 L / 8 D) in a plant incubator. At this time, when Agrobacteria were growing, 300 mg / L of carbenicillin was added to the co-cultivation liquid medium, washed, then dried on filter paper, and then re-inoculated on the medium.

[0104] Callus differentiated from the cuttings within 3 to 4 weeks after planting. Shoots were induced from the differentiated callus and when they grew to over 1 cm, they were transferred to rooting medium (Table 9) to induce roots. At this time, callus was removed as much as possible, and rooting was induced by transplanting the shoot parts. Once rooting was complete, the roots were washed with tap water to remove any remaining Agrobacteria in the roots, transferred to soil, and cultured while maintaining the incubation room environment and adapting to the external environment (humidity changes and non-sterile conditions).

[0105] FIG. 11 shows the changes in the objects according to the stages of plant differentiation from the callus.

[0106] As a result, as shown in Figures 12 to 14, tomato shoots transformed with D100 (Figure 12) or D121 (Figure 14) grew normally as plants, but tomato explants transformed with D120 did not grow normally (Figure 13).

[0107] Example 3. Sequencing of tomato plants generated by gene editing of DWF5, CPD and SMT1 To confirm the transformation of tomato plants obtained in Example 2, T7E1 assays (Figure 15) and Sanger sequencing (Figure 16 and Table 12) were performed on tomato controls transformed with D100 or D121.

[0108] Specifically, in the T7E1 assay, the target region was first amplified by PCR, and then the amplified PCR product was purified using a PCR purification kit. Heteroduplex formation was performed using the purified PCR product as a DNA template under the conditions in Table 11 and in the combinations shown in Table 10. Then, 1 μL of T7 endonuclease I (T7E1) was added and reacted at 37 ° C for 30 minutes. Finally, the activity of the T7E1 enzyme was inactivated by treating with 1 μL of proteinase K and reacting at 37 ° C for 5 minutes. Gene editing efficiency was evaluated by the degree of fragment concentration in the PCR product.

[0109] [Table 10]

[0110] [Table 11]

[0111] Sanger sequencing was performed as follows. The sgRNA target region was amplified from genomic DNA extracts using Q5 High-Fidelity DNA Polymerase (New England Biolabs) in a reaction volume of 20 μL. The PCR product was then cloned into a TA vector using All in one Cloning Kit (Biofact, South Korea), and 15–20 cloned clones per sample were sequenced individually. As a result, 14 tomato homozygotes with deleted DWF5 gene resulting from transformation with D100 were identified (Figure 15 and Figure 16). In addition, one tomato homozygote with deleted DWF5, CPD, and SMT1 genes transformed with D121 was identified (Figure 15 and Table 12). In the case of plants transformed with D121, the imbalance of plant hormones caused by gene deletion led to the phenomenon of no fruit formation.

[0112] [Table 12]

[0113] Example 4. Obtaining seeds and genetic phenotypic analysis of tomatoes produced by DWF5 gene editing Seeds (DWF5 homozygotes) obtained from seven tomatoes (T0 generation) in which the DWF5 gene was deleted by genetic scissors were sown and subjected to next-generation sequencing (NGS) to determine whether tomatoes in the next generation (T1) with the DWF5 gene deleted could be obtained.

[0114] As a result, it was confirmed that 12 of the 23 seeds (T1) obtained from the seven T0 subjects were homozygous (Figure 17). Furthermore, six seeds with 100% gene editing efficiency (indel efficiency) were selected from the above 12 seeds, and the occurrence of seeds (T2) and the amount of seeds obtained were confirmed. As a result, seeds were produced in five of the six subjects (Figure 18 and Table 13). Furthermore, no significant differences were observed in the phenotypes of the fruit, leaves, and stems of tomatoes in which the DWF5 gene was deleted compared to wild-type tomatoes. In this case, wild-type tomatoes (wild type, WT) were the control group.

[0115] [Table 13]

[0116] Example 5. Phenotypic analysis of T2 generation Example 5.1. Analysis of Vitamin D3 Precursor The seeds (T2) obtained in Example 4 were sown, and the content of provitamin D3 in the T2 generation tomatoes was measured by LC / MS. In this case, wild-type tomatoes (WT), the T2 generation of the #3-14 line (D100-3-14) (4 subjects), and the T2 generation of the #7-1 line (D100-7-1) (11 subjects) were used as samples. 7-dehydrocholesterol (provitamin D3, sigma, catalog 30800, lot BCBS6021), which is a precursor of animal provitamin D3, was used as a standard reagent. In addition, wild-type (WT) tomatoes were used as a control sample.

[0117] Specifically, a certain amount of each sample was weighed, and 30 mL of ethanol and 1 mL of 10% ethanolic pyrogallol were added. Then, 3 mL of 90% potassium hydroxide (KOH) was added and reacted in a water bath at 80°C for 30 minutes with vortexing every 10 minutes. After the reaction was completed, the mixture was cooled to room temperature, and then 30 mL of distilled water and 30 mL of n-hexane were added and mixed by shaking for 1 hour. Then, the supernatant was transferred to a separatory funnel, and 30 mL of n-hexane was added to the water layer and mixed by vortexing. The supernatant was transferred to a separatory funnel again, and 100 mL of distilled water was added to the hexane layer to wash with water. The washing process with water was repeated twice. Then, the hexane layer was dehydrated with anhydrous sodium sulfate, then concentrated, dissolved in 5 mL of methanol (MeOH), and filtered through a polytetrafluoroethylene (PTFE) filter to prepare the test solution (Figure 19).

[0118] The amount of 7-dehydrocholesterol contained in the test solution prepared by the above process was measured by LC / MS under the conditions shown in FIG.

[0119] As a result, 7-dehydrocholesterol was not detected in the fruit of WT tomatoes, but 7-dehydrocholesterol was detected in 4 objects in the T2 generation #3-14 line and 11 objects in the T2 generation #7-1 line (total of 15 objects) (Table 14, Figures 21 and 22).

[0120] [Table 14]

[0121] Example 5.2. Analysis of Vitamin D3 Precursors in Tomato Roots Seeds (T2) obtained in Example 4 were disinfected with 70% ethanol for 5 seconds and with 2% bleach for 20 minutes. The disinfected seeds were washed 4-5 times with sterile water and then placed on sterile filter paper to remove moisture. The seeds were sown on MS medium (Table 15) and cultured at 25°C under a 16-hour light / 8-hour dark cycle. After 2-3 weeks, when roots were formed, the roots were removed from the plate, and then the roots (hairy roots) were collected and analyzed for 7-DHC content by GC-MS method.

[0122] [Table 15]

[0123] GC-MS metabolic analysis was performed by mixing 10 mg of root with 3 mL of 0.1% ascorbic acid-ethanol and 0.05 mL of 5α-cholestane to obtain a root extract. The extract was mixed with 80% potassium hydroxide to proceed with saponification, and then mixed with hexane to separate the lipophilic substances. The separated lipophilic substances were derivatized using pyridine and N-methyl-N-trimethylsilyltrifluoroacetamide, followed by GC-MS (gas chromatography-quadrupole mass spectrometry) analysis. For quantitative and qualitative analysis, a calibration curve calculated from the standard substances was used.

[0124] As a result, 7-dehydrocholesterol was not detected in the roots of WT tomatoes, but 7-dehydrocholesterol was detected at concentrations of 1.3 μg / g and 1.0 μg / g in the T3 generation lines #3 (3-14-29) and #7 (7-1-15), respectively (Figure 28).

[0125] Example 5.3. Selection of antibiotic-free targets in the T2 generation In order to select targets without antibiotic resistance from the T2 generation #3-14 line (D100-3-14) and #7-1 line (D100-7-1) obtained in Example 4, the expression of antibiotic resistance genes in the mRNA of the targets was confirmed by RT-PCR.

[0126] As a result, 5 subjects of the #3-14 lineage (D100-3-14) and 11 subjects of the #7-1 lineage (D100-7-1) were confirmed to be subjects without antibiotic resistance (FIG. 23).

[0127] Among the subjects selected as those without antibiotic resistance, subjects #3-14-29 and #7-1-15 were checked for DWF5 gene editing.

[0128] As a result, as shown in Figures 24 and 25, the nucleotide sequence of the subject was different from that of the wild-type (WT) tomato (Figure 24), and since the sequence corresponding to exon 6 was deleted, it was confirmed that the amino acid sequence was also different from that of the wild-type (WT) tomato (Figure 25).

[0129] Example 5.4. Confirmation of T2 Generation Phenotype and Seed Production The T2 generation #3-14 line (D100-3-14) and #7-1 line (D100-7-1) obtained in Example 4 were checked for phenotype and seed production. Specifically, when only the DWF5 gene (DWF5-1, SEQ ID NO: 63), which is mainly expressed in fruit, was deleted, no significant difference was observed in the phenotype of fruit, leaves and stems compared to wild-type tomatoes (FIG. 27). Therefore, in the present invention, by deleting only the DWF5-1 (SEQ ID NO: 63) gene, tomatoes that can induce the concentration of provitamin D3 while complementing the problem of seed production could be produced.

Claims

1. A transformed tomato containing high levels of 7-dehydrocholesterol of formula 1 below. 【Chemistry 1】

2. 2. The transformed tomato of claim 1, wherein 7-dehydrocholesterol is found in high concentrations in the fruits and roots of the tomato.

3. 2. The transformed tomato of claim 1, wherein the concentration of 7-dehydrocholesterol is 0.01 mg to 1.5 mg per 100 g of weight.

4. 2. The transformed tomato of claim 1, which has been genetically engineered to reduce expression or activity of the DWF5-1 gene or DWF5-1 protein as compared to a wild-type tomato.

5. 5. The transformed tomato of claim 4, wherein the DWF5-1 gene comprises the nucleic acid sequence of SEQ ID NO:

63.

6. 5. The transformed tomato of claim 4, wherein said genetic engineering is induced by a modification in the nucleic acid sequence of said DWF5-1 gene.

7. 7. The transformed tomato of claim 6, wherein said modification of said nucleic acid sequence comprises a sequence which differs from that of said wild-type tomato by a substitution, deletion or insertion.

8. 5. The transformed tomato of claim 4, which has been further genetically engineered to reduce expression or activity of a CPD gene or CPD protein.

9. 9. The transformed tomato of claim 8, wherein the CPD comprises the nucleic acid sequence of SEQ ID NO:

5.

10. 9. The transformed tomato of claim 8, wherein the genetic engineering is induced by a modification in the nucleic acid sequence of the CPD gene.

11. 9. The transformed tomato of claim 8, further genetically engineered to reduce expression or activity of the SMT1 gene or SMT1 protein.

12. 12. The transformed tomato of claim 11, wherein the SMT1 comprises the nucleic acid sequence of SEQ ID NO:

7.

13. 12. The transformed tomato of claim 11, wherein said genetic engineering is induced by a modification in the nucleic acid sequence of the SMT1 gene.

14. The transformed tomato of claim 4 , which is homozygous.

15. 5. The transformed tomato of claim 4, which produces seeds.

16. A vector comprising at least one sgRNA (single guide RNA) that binds complementarily to a nucleotide sequence of the DWF5-1 gene and a nucleotide sequence encoding a CRISPR (clustered regularly interspaced palindromic repeats) associated protein.

17. 17. The vector of claim 16, wherein the sgRNA comprises any one of the nucleotide sequences selected from the group consisting of SEQ ID NO: 20, SEQ ID NO: 21, and combinations thereof.

18. 17. The vector of claim 16, further comprising one or more sgRNAs that bind complementary to a nucleotide sequence of the CPD gene.

19. 20. The vector of claim 18, wherein the sgRNA comprises any one of the nucleotide sequences selected from the group consisting of SEQ ID NO:28, SEQ ID NO:29, and combinations thereof.

20. 17. The vector of claim 16, further comprising one or more sgRNAs that bind complementary to a nucleotide sequence of the SMT1 gene.

21. 21. The vector of claim 20, wherein the sgRNA comprises any one of the nucleotide sequences selected from the group consisting of SEQ ID NO: 32, SEQ ID NO: 33, and combinations thereof.

22. The CRISPR-related proteins include Cas9, Cpf1, c2c1, C2c2, Cas13, c2c3, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7. , Cas8a, Cas8a1, Cas8a2, Cas8b, Cas8c, Csn1, Csx12, Cas10, Cas10d, Cas10, Cas10d, CasF, CasG, CasH, Csyl, Csy2, Csy3, Cse1 (CasA), Cs The vector of claim 16, which is any one selected from the group consisting of e2 (CasB), Cse3 (CasE), Cse4 (CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csxl, Csx15, Csfl, Csf2, Csf3, Csf4 and Cul966.

23. 23. The vector of claim 22, wherein the CRISPR associated protein is Cas9.

24. A method for producing a transformed tomato containing a high concentration of 7-dehydrocholesterol, comprising introducing the vector according to any one of claims 16 to 23 into tomato using Agrobacterium.

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