KR102550308B1 - Method for producing genome-edited tomato plant with increased salt tolerance by SlHKT1;2 gene editing and genome-edited tomato plant with increased salt tolerance produced by the same method - Google Patents

Abstract

The present invention relates to a method for producing a genome-corrected tomato plant with increased salt tolerance by SlHKT1;2 ( Solanum lycopersicum High-affinity K ⁺ transporter 1;2) gene correction and a genome-corrected tomato plant with increased salt tolerance prepared by the method In this regard, in the present invention, a tomato with increased salt tolerance was developed through a single amino acid conversion (N217D) of the HKT1 ;2 protein by inducing a specific mutation in the tomato HKT1;2 gene. Since the tomato with increased salt tolerance according to the present invention shows an excellent germination rate in high salt conditions, it can be used as a tomato applicable to cultivation of salty tomatoes or high salt conditions.

Description

SlHKT1; 2 Method for producing genome-edited tomato plants with increased salt tolerance by gene editing and genome-edited tomato plants with increased salt tolerance prepared by the method {Method for producing genome-edited tomato plant with increased salt tolerance by SlHKT1; 2 gene editing and genome-edited tomato plant with increased salt tolerance produced by the same method}

The present invention relates to a method for producing a genome-corrected tomato plant with increased salt tolerance by SlHKT1;2 ( Solanum lycopersicum High-affinity K ⁺ transporter 1;2) gene correction and a genome-corrected tomato plant with increased salt tolerance prepared by the method it's about

New breeding technology using genetic scissors technology is a technology that secures new varieties with modified characteristics even though there is no externally introduced gene in the finally developed plant. It is attracting attention as a technology that can avoid or mitigate the safety controversy for GM (genetically-modified) crops.

Commercialization of genome editing technologies using homologous recombination (HR) or homology-directed DNA repair (HDR) in plants has been an area considered the holy grail of plant biotechnology. The fact that double strand break (DSB) increases HDR in Nicotiana species plants was reported in the early 1990s, and genetic scissors or genome editing/editing technology to modify the genome by inducing DSB have been reported in 20 Although known years ago, the use of genome editing/editing techniques in plant systems has been very limited. The present inventors have reported a method for increasing the efficiency of gene editing based on homologous recombination in plants through previous studies (Korean Patent Registration No. 2002443), and in the present invention, the above technology was applied to tomato, a major fruit and vegetable, to improve traits. We tried to develop genome-edited tomato plants.

On the other hand, Korean Patent Publication No. 2017-0140922 discloses 'OsSIRP1 gene derived from rice that increases salt tolerance of plants and its use' using salt-induced RING Finger Protein, and Korean Patent Publication No. 2009-0041648 discloses ' A method for increasing the salt tolerance of a plant by overexpressing the SyFBP/SBpase gene derived from cyanobacteria has been disclosed, but the method for producing a genome-corrected tomato plant with increased salt tolerance by the SlHKT1;2 gene correction of the present invention and the method prepared by the method Genome-edited tomato plants with increased salt tolerance have not been described.

The present invention was derived from the above needs, and the present inventors prepared a genome-edited tomato plant in which a single amino acid mutation (N217D) was induced in the HKT1;2 protein of tomato using a homologous recombination-based gene editing technology. The present invention was completed by confirming that the genome-edited tomato plants showed excellent germination rate and growth characteristics in high salt conditions, although there was no difference in morphologically from genome-free tomato plants.

In order to solve the above problems, the present invention between the LB (left border) and RB (right border) sequences of the Ti plasmid, the antibiotic marker expression cassette; A donor sequence including a template sequence for homology-directed DNA repair (HDR) of HKT1;2 (High-affinity K ⁺ transporter 1;2) gene derived from tomato ( Solanum lycopersicum ); dual HKT1;2 crRNA expression cassette; And an endonuclease (endonuclease) expression cassette; provides a method for producing a genome-edited tomato plant with increased salt tolerance, comprising the step of transforming tomato plant cells with a recombinant vector comprising a.

In addition, the present invention provides a genome-corrected tomato plant with increased salt tolerance prepared by the above method and a genome-corrected seed thereof.

In addition, the present invention is an antibiotic marker expression cassette between the LB and RB sequences of the Ti plasmid; a donor sequence including a template sequence for homology-based DNA repair of tomato-derived HKT1;2 gene; dual HKT1;2 crRNA expression cassette; And an endonuclease expression cassette; It provides a composition for genome editing for increasing the salt tolerance of tomato plants, containing a recombinant vector containing as an active ingredient.

The salt tolerance of the plant is an agronomic characteristic necessary for improving the quality of agricultural products and cultivating in high-salt soil, and the salt-tolerant tomato plant prepared by the method according to the present invention can be used as a salty tomato seed, It can be usefully used in the cultivation of conditions.

Figure 1 shows a CRISPR/Cpf1-based replicon system for HKT1;2 ^N217D editing through homology-directed DNA repair (HDR), and (a) is Sanger sequencing of HDR event #C156 in which the HKT1;2 gene is corrected. (b) is a schematic diagram of the HDR construct for HKT1;2 gene correction, and (c) is the HDR event #C156 plant in which the HKT1;2 gene was corrected, and the developmental phenotype under normal conditions is wild type (WT)- It is a photograph showing that there is no difference compared to similar event plants.
FIG. 2 shows a donor sequence (corresponding to the HKT1;2 donor in FIG. 1B, SEQ ID NO: 2) including a template sequence for HDR of the tomato-derived HKT1;2 gene. Bases marked in red are positions where asparagine (N)-coding bases are modified to aspartic acid (D)-coding bases, and fluorescently marked in yellow are bases at PAM sites modified to prevent recognition and cleavage by LbCpf1-gRNA. show the location of
Figure 3 shows the results of germination tests of HDR event #C156 plants and wild-type plants in which the HKT1;2 gene was corrected under high salt conditions (100 mM NaCl).

In order to achieve the object of the present invention, the present invention between the LB (left border) and RB (right border) sequences of the Ti plasmid, the antibiotic marker expression cassette; A donor sequence including a template sequence for homology-directed DNA repair (HDR) of HKT1;2 (High-affinity K ⁺ transporter 1;2) gene derived from tomato ( Solanum lycopersicum ); dual HKT1;2 crRNA expression cassette; And an endonuclease (endonuclease) expression cassette; provides a method for producing a genome-edited tomato plant with increased salt tolerance, comprising the step of transforming tomato plant cells with a recombinant vector comprising a.

In the production method of the present invention, the template sequence for HDR of the tomato-derived HKT1;2 gene is the amino acid sequence of SEQ ID NO: 5, and the 217th asparagine (N) in the tomato HKT1;2 protein sequence is aspartic acid (aspartic acid). It may include a sequence in which the first base of the asparagine coding base (AAT) is changed to guanine (G) so that it can be substituted with acid, D), and the position of the base changed to G is of SEQ ID NO: 2 It is the 1,015th base in the donor sequence consisting of base sequences (see FIG. 2).

In the production method according to the present invention, the antibiotic marker expression cassette, donor sequence, crRNA expression cassette, and endonuclease expression cassette are composed of the nucleotide sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, respectively. It may be, but is not limited thereto.

In the present invention, the term "expression cassette" includes a promoter and a target protein coding sequence to be expressed or a polynucleotide to be expressed, and a unit cassette capable of expressing a target protein or target sequence to produce a target protein or target sequence. means

The antibiotic marker expression cassette consisting of the nucleotide sequence of SEQ ID NO: 1 was cloned from pICSL11024 (pICH47732::NOSp-NPTII-OCST; Addgene Plasmid #51144), and the 1st to 354th bases are NOS promoter and TMV Ω, 355 to 1,149 The 1st base is the NptII coding sequence, and the 1,150th to 1,885th base is the OCS (octopine synthase) terminator sequence.

In addition, the crRNA expression cassette composed of the nucleotide sequence of SEQ ID NO: 3 consists of the following configuration: bases 1 to 75 are AtU6 promoters, bases 76 are transcription start bases (G), bases 77 to 97 are LbCpf1 scaffold, bases 98 to 120 are LbCpf1_gHKT1;2.1, bases 121 to 141 are LbCpf1 scaffold, bases 142 to 164 are LbCpf1_gHKT1;2.2, bases 165 to 170 are termination signals.

In addition, the endonuclease expression cassette composed of the nucleotide sequence of SEQ ID NO: 4 consists of the following configuration: CaMV 35S promoter sequence in which the AtUBQ1 intron sequence (bases 1,350 to 1,653) is inserted in the 3' terminal direction ( bases 1 to 1,349 and 1,654 to 1,736), SV40 NLS sequence located before (bases 1,737 to 1,757) and after (bases 5,654 to 5,674 and 5,681 to 5,701) of the LbCpf1 coding sequence, Trp1 intron in the 5'-end direction The LbCpf1 coding sequence (bases 1,812 to 5,605) with the sequence (bases 1,872 to 1,981) inserted, and the HA tag (bases 5,609 to 5,635) and a linker sequence.

As used herein, the term 'genome/gene editing' refers to a technology capable of introducing target-directed mutations into genomic sequences of animal and plant cells, including human cells, and refers to one or more nucleic acid molecules by cutting DNA. It refers to a technology that can knock-out or knock-in a specific gene by deletion, insertion, substitution, etc., or introduce mutations into non-coding DNA sequences that do not produce proteins. . For the purposes of the present invention, the genome editing may be to introduce mutations into plants using an endonuclease, such as Cpf1 protein and guide RNA.

In the production method according to one embodiment of the present invention, the endonuclease protein is Cas9 (CRISPR associated protein 9), Cpf1 (CRISPR from Prevotella and Francisella 1), TALEN (Transcription activator-like effector nuclease), ZFN ( Zinc Finger Nuclease) or one or more functional analogues thereof, preferably LbCpf1 (Lachnospiraceae bacterium ND2006 Cpf1) protein, but is not limited thereto. Genetic information of the endonuclease can be obtained from a known database such as GenBank of National Center for Biotechnology Information (NCBI).

In the production method according to the present invention, the method of transducing the recombinant vector into plant cells is the calcium/polyethylene glycol method for protoplasts (Krens et al., 1982, Nature 296: 72-74; Negrutiu et al., 1987 , Plant Mol. Biol. 8: 363-373), electroporation of protoplasts (Shillito et al., 1985, Bio/Technol. 3: 1099-1102), microinjection into plant elements (Crossway et al., 1986 , Mol. Gen. Genet. 202: 179-185), particle bombardment of various plant elements (DNA or RNA-coated) (Klein et al., 1987, Nature 327: 70), Agrobacterium tumefaciens ( Agrobacterium tumefaciens ) in (incomplete) virus mediated gene transfer (EP 0 301 316) and the like.

The "plant cell" used for the transformation can 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, i.e., single cells, protoplasts. (protoplast), shoot and callus tissue. The plant tissue may be in planta or may be in organ culture, tissue culture or cell culture. Preferred plant cells according to the present invention are explants.

In the production method of the present invention, any method known in the art may be used for regenerating a genome-corrected tomato plant from tomato plant cells transformed with the recombinant vector and having the HKT1;2 genome corrected.

The present invention also provides a genome-corrected tomato plant with increased salt tolerance prepared by the above method and a genome-corrected seed thereof.

The genome-edited tomato plants with increased salt tolerance according to the present invention are those in which the 217th amino acid of the HKT1;2 protein, which plays an important role in maintaining K ⁺ absorption under salt stress conditions, is substituted from N to D, and homology-based DNA repair (homology -Directed DNA repair (HDR)-based gene editing technology is used to correct the HKT1;2 gene, and it is a genome-edited tomato plant with increased germination rate and growth characteristics in high salt conditions compared to tomato plants without genome correction. .

The present invention also relates to an antibiotic marker expression cassette between the LB (left border) and RB (right border) sequences of the Ti plasmid; A donor sequence including a template sequence for homology-directed DNA repair (HDR) of a tomato-derived HKT1;2 (High-affinity K ⁺ transporter 1;2) gene; dual HKT1;2 crRNA expression cassette; And an endonuclease (endonuclease) expression cassette; to provide a genome editing composition for increasing the salt tolerance of tomato plants containing a recombinant vector containing as an active ingredient.

The composition for genome editing of the present invention is a recombinant vector containing a donor sequence (SEQ ID NO: 2) including an HDR template sequence whose base is modified so that asparagine at position 217 of the tomato HKT1;2 protein can be substituted with aspartic acid. When tomato plant cells are treated with the composition as an active ingredient, the HKT1; 2 ^N217D mutation can be induced through correction of the HKT1;2 gene, thereby increasing the salt tolerance (high salt resistance) of tomato plants. It can.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are only to illustrate the present invention, and the content of the present invention is not limited to the following examples.

Materials and Methods

1. Construction and cloning of HDR test system

Overall design principles and all cloning procedures follow the MoClo (Weber et al., (2011) PLoS ONE, 6, e16765) and Golden Gate (Engler et al., (2014) ACS Synth. Biol. 3, 839-843) protocols. Followed. pHRHKT12.1 was designed by amplifying the long intergenic region (LIR) and short intergenic region (SIR) of the pLSLR plasmid (Cermak et al., (2015) Genome Biol. 16, 232). A level 2 Golden Gate BpiI restriction site was incorporated into the replicon to replicate the HDR expression cassette. Release of the circular DNA replicon was verified in tomato leaf and tomato cotyledon explants. The pTC147 and pTC217 plasmids were obtained from Addgene and used as references. The LbCpf1-based HDR replicon contains two guide RNAs (LbCpf1_gRNA1 and LbCpf1_gRNA2). Donor DNA was constructed via insertion of the CaMV 35S promoter to induce overexpression. A dual guide RNA structure was designed along the 23-nt guide RNA sequence after multiplexing LbCpf1 crRNA as a tandem repeat of scaffold RNA. The crRNA was induced by the AtU6 promoter (Kamun Lab, Addgene #46968) (Figures 1a and 1b).

2. Tomato Transformation

In the present invention, tomato (Hongkwang cultivar) was used as a model crop for HDR enhancement. All binary vectors were transformed into Agrobacterium tumefaciens strain GV3101 using electroporation, and Agrobacterium-mediated transformation was used to transfer the editing tool to tomato cotyledon fragments. Explants for transformation were prepared using 7-day-old tomato cotyledons. Sterilized seeds of the Honggwang cultivar were planted on MSO medium (half-strength MS medium containing 30 g/L sucrose, pH 5.8) and cultured under the conditions of a 16-hour light/8-hour dark photoperiod and a temperature of 25±2°C. did After collecting 7-day-old seedlings and cutting cotyledons into 0.2-0.3 cm-sized fragments, the fragments (= explants) were mixed with PREMC medium (MS basal salts, Gamboorg B5 vitamins, 2.0 mg/L of Zeatin trans isomer, 0.2 mg /L of indolyl acetic acid, 1 mM of putrescine and 30 g/L of glucose, pH 5.7) for 1 day. The pre-cultured explants were punctured to make a small hole and then co-cultured with Agrobacterium having the HR construct. Thereafter, the transformed explants were selected through a selection medium containing 300 mg/L of timementin and 80 mg/L of kanamycin. The detailed method is described in detail in Butien Van et al. (Plant Biotechnol J. 2020 Mar 16. doi: 10.1111/pbi.13373.).

3. HDR event evaluation

Evaluation of gene-targeting junctions was performed by PCR and Sanger using primers on the left (UPANT1-F1/NptII-R1) and right (ZY010F/TC140R) junction flanks and high-performance Taq DNA polymerase (Phusion Taq, Thermo Fisher Scientific, USA). It was performed through Sanger sequencing. The levels of DNA amplicons and associated donor templates were evaluated using semi-quantitative PCR and quantitative PCR (KAPA SYBR FAST qPCR Kits, Sigma-Aldrich, USA), respectively. The genetic pattern of the HDR-edited allele was analyzed by PCR and Sanger sequencing using genome-edited generation 1 (GE1). Circular replicon was detected using primers for pHR01, multiple replicon or pTC217. The primer information is described in Butienban et al. (Plant Biotechnol J. 2020 Mar 16. doi: 10.1111/pbi.13373.).

Example 1. HKT1;2 Preparation of gene-edited tomato plants

High-affinity K ⁺ transporter 1;2 (hereafter HKT1;2) plays an important role in maintaining K ⁺ uptake under salt stress conditions. Salt tolerance is determined by a single asparagine (N)/aspartic acid (D) mutation in the pore region of HKT1;2.

The present inventors use a CRISPR/Cpf1-based replicon system (refer to Korean Patent Registration No. 2002443) using a donor template of the HKT1;2 gene that does not contain an antibiotic selection marker or color marker associated with the allele. Thus, gene editing of tomato plants was attempted. As a result of analyzing 150 events, we succeeded in generating a heterogeneous but perfect HDR GE0 event (#C156) to produce a salt tolerance allele (N217D). Event #C156 with the HKT1;2 ^N217D allele showed a normal phenotype compared to the wild type and fruited normally.

#C156 plants were self-pollinated to produce GE1 generation plants, and the seeds of the plants were seeded in a medium containing 100 mM sodium chloride, and their germination rate and growth characteristics were compared with those of the wild type. As a result, as shown in FIG. 3 , the GE1 generation seeds of event #C156 having the HKT1;2 ^N217D allele showed higher germination rates and growth characteristics compared to the wild type even under high salt conditions.

<110> INDUSTRY-ACADEMIC COOPERATION FOUNDATION GYEONGSANG NATIONAL UNIVERSITY <120> Method for producing genome-edited tomato plant with increased salt tolerance by SlHKT1;2 gene editing and genome-edited tomato plant with increased salt tolerance produced by the same method <130> PN20194 <160> 5 <170> KoPatentIn 3.0 <210> 1 <211> 1885 <212> DNA <213> Artificial Sequence <220> <223> selection marker expression cassette <400> 1 attcggatcc ggagcggaga attaagggag tcacgttatg acccccgccg atgacgcggg 60 a caagccgtt ttacgtttgg aactgacaga accgcaacgt tgaaggagcc actgagccgc 120 gggtttctgg agtttaatga gctaagcaca tacgtcagaa accattattg cgcgttcaaa 180 agtcgcctaa ggtcactatc agctagcaaa tatttcttgt caaaaatgct ccactgacgt 240 tccataaatt cccct cggta tccaattaga gtctcatatt cactctccta tttttacaac 300 aattaccaac aacaacaaac aacaaacaac attacaatta catttacaat taccatggtt 360 gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat 420 gactgggcac aacagacaat cggctgct ct gatgccgccg tgttccggct gtcagcgcag 480 gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaggac 540 gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac 600 gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc 660 ctgtcatctc acct tgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg 720 ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag 780 cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat 840 caggggctcg cgccagccga actgttcgcc aggctcaagg cgcgcatgcc cgacggcgag 900 gatctcgtcg tgactcatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc 960 ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg 1020 ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg 1080 ctttacggta tcgccg ctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag 1140 ttcttctgag cgggactctg gggttcgcta gagtcctgct ttaatgagat atgcgagacg 1200 cctatgatcg catgatattt gctttcaatt ctgttgtgca cgttgtaaaa aacctgagca 1260 tgtgtagctc agatccttac cgccggtttc ggttcattct aatgaatata tcacccgtta 1320 ctatcgtatt tttatgaata atattctccg ttcaatttac tgattgtacc a aatttcaaa 1560 agtgccccag gggctagtat ctacgacaca ccgagcggcg aactaataac gctcactgaa 1620 gggaactccg gttccccgcc ggcgcgcatg ggtgagattc cttgaagttg agtattggcc 1680 gtccgctcta ccgaaagtta cgggcaccat tcaaccc ggt ccagcacggc ggccgggtaa 1740 ccgacttgct gccccgagaa ttatgcagca tttttttggt gtatgtgggc cccaaatgaa 1800 gtgcaggtca aaccttgaca gtgacgacaa atcgttgggc gggtccaggg cgaattttgc 1860 gacaacatgt cgaggctcag ccgct 1885 <210> 2 <211> 1851 <212> DNA <213> Artificial Sequence <220> <223> donor sequence <400> 2 ggagactcat gaacttgaaa tgcatagaat gcctatttta acacgttttt aacaaataat 60 aaataagttg tatagcatct tataattcca ctaactctat gataaggaaa tatcagtgat 120 catttggcaa gaattattat attctaactc cttttggtac gtaccatatg ttgttgggat 180 taatttaaat cattggtaag aattattata ttctaactcc ttttgatacg taccatatgt 240 tattgggatt aatttaaagc tta cacaatg tgtctagtat aggcattcca ctacacaatt 300 tttactataa attgaaccct tgagtattat aataaaactt aagagacaaa caaatttatt 360 ttactaatga agtcatcact ttcaatttct ttctttaaga ttatgatgag tcttagggta 420 aagccatttt ggattgaatt agggtattt c acaacccttt cattgttagg tttcttagct 480 ttgaattatg tttcaaaacc tagaacccta ccgtcttttc gtcctcaaaa cctagatgtt 540 cttttcactt ctgtttcttc aaccacagtt tctagtatgt ccactattga aatggaagtt 600 ttctcaaatg ttcaacttgt tttcatgacc atattgatgt ttcttggtgg ggaagctttt 660 acctcttt tc ttagccttaa actcatcaag aataaagaaa gcaaagataa atcttttagt 720 aacaaagatt atgagctagg gaatgtaata aacgttgaca ataagttaga agatgtgata 780 ataataaacc ctatcgaaga tcatattcat gatcatcacg atgaaattat aaagattaaa 840 tcgataaaat tgttgag taa tgtggttttt ggatatattc ttgttgttat tcttcttggt 900 tcatcgttag tttctctcta tataataatc atccctagcg ccaaacaaat ccttgaccaa 960 aaaggcctta atttacatac tttctctcta ttcaccacag tatcaacttt tgcagattgt 1020 ggtttcttac caacaaatga aaacatgatg attttcaaga aaaattcagg acttcttctc 1080 attcttatcc ctcaagtcct tctagg gaac actttgtttg ctccttgttt acgcatcgtt 1140 ataatgttct tatggaaaat cacaaagaga catgagtatg agtatatttt gaagaactca 1200 aaatgtgttg gatattcaca tatttttcca agttatgaaa caattggtat tgctattact 1260 gttgtgggat tatagtatt tcaatttgtt atgttttgtt cattggagtg gaattctgaa 1320 ggtacttctg gattaagtac ttatgagaag attgtgggat ctttgtttga agttgtgaat 1380 acaaggcatg ctggtctatc tgtatttgat ctttcaactt ttactccatc aatcttagta 1440 ttgtttgcct taatgatgta agtattctct acaatctatg ttctttcgtg ttctgtctca 1500 atttacgtga catatatact acttctttgt tttatatct g tgtctctgga agaattaatg 1560 tcaactttct atagttagaa ataacttaat tttcaaattc cctttttatc tttgattcac 1620 atgaatatct aagatttgct ttatattgca aattttcaaa tttgatgtta agttattaaa 1680 aagtatcaca tgaacaagag agaggaga aa gtagttcatt gtttagaaat ttatcatcca 1740 catgcaacta tgttaatact aacatatttg tttaagttgt tgtaatagga tatgataaac 1800 ttttaacaac aaattaagtt taactattaa acaaatgtgt taaaagtgtt a 1851 <210> 3 <211> 170 <212> DNA <213> Artificial Sequence <220> <223> crRNA expression cassette <400> 3 tgatcaaaag tcccacatcg atcaggtgat atatagcagc ttagtttata taatgataga 60 gtcgacatag cgattgtaat ttctactaag tgtagatact attcaccaca gtatcaactt 120 taatttctac taagtgtaga tcctacaaat gaaaacatga tgattttttt 170 <210> 4 <211> 5934 <212> DNA <213> Artificial Sequence <220> <223> endonuclease expression cassette <400> 4 gaattccaat cccacaaaaa tctgagc tta acagcacagt tgctcctctc agagcagaat 60 cgggtattca acaccctcat atcaactact acgttgtgta taacggtcca catgccggta 120 tatacgatga ctggggttgt acaaaggcgg caacaaacgg cgttcccgga gttgcacaca 180 agaaatttgc cactattaca gaggcaagag cagcagctga cgcgtacaca acaagtcagc 240 aaacagacag gttgaacttc atccccaaag gagaagctcacaactccc aagagctttg 300 ctaaggccct aacaagccca ccaaagcaaa aagcccactg gctcacgcta ggaaccaaaa 360 ggcccagcag tgatccagcc ccaaaagaga tctcctttgc cccggagatt acaatggacg 420 atttcctcta tctttacgat ctaggaagga agttcgaagg tgaaggtgac gacactatgt 480 tcaccactga taatgagaag gttagcctct tcaatttcag aaagaatgct gacccacaga 72 0 cgattcaagg cttgcttcat aaaccaaggc aagtaataga gattggagtc tctaaaaagg 780 tagttcctac tgaatctaag gccatgcatg gagtctaaga ttcaaatcga ggatctaaca 840 gaactcgccg tcaagactgg cgaacagttc atacagagtc ttttacgact caatga caag 900 aagaaaatct tcgtcaacat ggtggagcac gacactctgg tctactccaa aaatgtcaaa 960 gatacagtct cagaagatca aagggctatt gagacttttc aacaaaggat aatttcggga 1020 aacctcctcg gattccattg cccagctatc tgtcacttca tcgaaaggac agtagaaaag 1080 gaaggtggct cctacaaatg ccatcattgc gataaaggaa aggctatcat tcaagatctc 11 40 tctgccgaca gtggtcccaa agatggaccc ccacccacga ggagcatcgt ggaaaaagaa 1200 gaggttccaa ccacgtctac aaagcaagtg gattgatgtg acatctccac tgacgtaagg 1260 gatgacgcac aatcccacta tccttcgcaa gacccttcct ctata taagg aagttcattt 1320 catttggaga ggacacgctc gagtataagg taaatttctg tgttccttat tctctcaaaa 1380 tcttcgattt tgttttcgtt cgatcccaat ttcgtatatg ttctttggtt tagattctgt 1440 taatcttaga tcgaagatga ttttctgggt ttgatcgtta gatatcatct taattctcga 1500 ttagggtttc atagatatca tccgatttgt tcaaataatt tgagtatatttgt cgaatta 1560 ctcttcgatt tgtgatttct atctagatct ggtgttagtt tctagtttgt gcgatcgaat 1620 ttgtcgatta atctgagttt ttctgattaa caggagctca tttttacaac aattaccaac 1680 aacaacaaac aacaaacaac attacaatta catttacaat tatcgataca atgatg ccca 1740 agaagaagcg caaggtggga cgcgtctgca ggatatcaag cttgcggtac cgcgggcccg 1800 ggatcgccac catgagcaag ctggagaagt ttacaaactg ctactccctg tctaagaccc 1860 tgaggttcaa ggtaaagcct cgatttttgg gtttaggtgt ctgcttatta gagtaaaaaac 1920 acatcctttg aaattgtttg tggtcatttg attgtgctct tgatccattg aattgctgca 1980 ggccatccct gtgggcaaga cccaggagaa catcgacaat aagcggctgc tggtggagga 2040 cgagaagaga gccgaggatt ataagggcgt gaagaagctg ctggatcgct actatctgtc 2100 ttttatcaac gacgtgctgc acagcatcaa gctgaagaat ctgaacaatt acatcagcct 2160 gt tccggaag aaaaccagaa ccgagaagga gaataaggag ctggagaacc tggagatcaa 2220 tctgcggaag gagatcgcca aggccttcaa gggcaacgag ggctacaagt ccctgtttaa 2280 gaaggatatc atcgagacaa tcctgccaga gttcctggac gataaggacg agatcgccct 2340 ggtgaacagc ttcaatggct ttaccacagc cttcaccggc ttctttgata acagagagaa 2400 tatgttttcc gag gaggcca agagcacatc catcgccttc aggtgtatca acgagaatct 2460 gacccgctac atctctaata tggacatctt cgagaaggtg gacgccatct ttgataagca 2520 cgaggtgcag gagatcaagg agaagatcct gaacagcgac tatgatgtgg aggatttctt 2580 tgaggg cgag ttctttaact ttgtgctgac acaggagggc atcgacgtgt ataacgccat 2640 catcggcggc ttcgtgaccg agagcggcga gaagatcaag ggcctgaacg agtacatcaa 2700 cctgtataat cagaaaacca agcagaagct gcctaagttt aagccactgt ataagcaggt 2760 gctgagcgat cgggagtctc tgagcttcta cggcgagggc tatacatccg atgaggaggt 2820 gctggaggtg tttagaaaca ccctgaacaa gaac agcgag atcttcagct ccatcaagaa 2880 gctggagaag ctgttcaaga attttgacga gtactctagc gccggcatct ttgtgaagaa 2940 cggccccgcc atcagcacaa tctccaagga tatcttcggc gagtggaacg tgatccggga 3000 caagtggaat gccgagtatg acgatat cca cctgaagaag aaggccgtgg tgaccgagaa 3060 gtacgaggac gatcggagaa agtccttcaa gaagatcggc tccttttctc gc catcatga aggacctgct 3300 ggattctgtg aagagcttcg agaattacat caaggccttc tttggcgagg gcaaggagac 3360 aaacagggac gagtccttct atggcgattt tgtgctggcc tacgacatcc tgctgaaggt 3420 ggaccacatc tacgatgcca tccgcaatta tgtgacccag aagccctact ctaaggataa 3480 gttcaagctg tattttcaga accctcagtt catgggcggc tgggacaagg ataaggagac 3540 agactatcgg gccaccatcc tgagatacgg ctccaagtac tatctggcca tcatggataa 3600 gaagtacgcc aagtgcctgc agaagatcga caaggacgat gtgaacggca attacgagaa 3660 gatcaactat aagctgctgc ccggccctaa taagatgctg ccaaaggt gt tcttttctaa 3720 gaagtggatg gcctactata accccagcga ggacatccag aagatctaca agaatggcac 3780 attcaagaag ggcgatatgt ttaacctgaa tgactgtcac aagctgatcg acttctttaa 3840 ggatagcatc tcccggtatc caaagtggtc caatgcctac gatttcaact tttctgagac 3900 agagaagtat aaggacatcg ccggctttta cagagaggtg gaggagcagg gctataaggt 3960 gagcttcgag tctgccagca agaaggaggt ggataagctg gtggaggagg gcaagctgta 4020 tatgttccag atctataaca aggacttttc cgataagtct cacggcacac ccaatctgca 4080 caccatgtac ttcaagctgc tgtttgacga gaacaatcac ggacagatca ggctgagc gg 4140 aggagcagag ctgttcatga ggcgcgcctc cctgaagaag gaggagctgg tggtgcaccc 4200 agccaactcc cctatcgcca acaagaatcc agataatccc aagaaaacca caaccctgtc 4260 ctacgacgtg tataaggata agaggttttc tgaggaccag tacga gctgc acatcccaat 4320 cgccatcaat aagtgcccca agaacatctt caagatcaat acagaggtgc gcgtgctgct 4380 gaagcacgac gataacccct atgtgatcgg catcgatagg ggcgagcgca atctgctgta 4440 tatcgtggtg gtggacggca agggcaacat cgtggagcag tattccctga acgagatcat 4500 caacaacttc aacggcatca ggatcaagac agattaccac tctctgctgg acaagaagga 4560 gaaggagagg ttcgaggccc gccagaactg gacctccatc gagaatatca aggagctgaa 4620 ggccggctat atctctcagg tggtgcacaa gatctgcgag ctggtggaga agtacgatgc 4680 cgtgatcgcc ctggaggacc tgaactctgg ctttaagaat agccgcgtga aggtggagaa 4740 gcagg tgtat cagaagttcg agaagatgct gatcgataag ctgaactaca tggtggacaa 4800 gaagtctaat ccttgtgcaa caggcggcgc cctgaagggc tatcagatca ccaataagtt 4860 cgagagcttt aagtccatgt ctacccagaa cggcttcatc ttttacatcc ctgcctggct 4920 gacatccaag atcgatccat ctaccggctt tgtgaacctg ctgaaaacca agtataccag 4980 catcgccgat tccaagaagt tcatcagctc ctttgacagg atcatgtacg tgcccgagga 5040 ggatctgttc gagtttgccc tggactataa gaacttctct cgcacagacg ccgattacat 5100 caagaagtgg aagctgtact cctacggcaa ccggatcaga atcttccgga atcctaagaa 5160 gaacaacgtg ttcgactggg aggaggtgtg cctgaccagc gcctataagg agctgttcaa 5220 caagtacggc atcaattatc agcagggcga tatcagagcc ctgctgtgcg agcagtccga 5280 caaggccttc tactctagct ttatggccct gatgagcctg atgctgcaga tgcggaacag 5340 catcacaggc cgcaccgacg tggattttct gatcagccct gtgaagaact ccgacggcat 5400 cttctacgat agcc ggaact atgaggccca ggagaatgcc atcctgccaa agaacgccga 5460 cgccaatggc gcctataaca tcgccagaaa ggtgctgtgg gccatcggcc agttcaagaa 5520 ggccgaggac gagaagctgg ataaggtgaa gatcgccatc tctaacaagg agtggctgga 5580 gtacgcc cag accagcgtga agcacgccta tccctatgac gtgcccgatt atgccagcct 5640 gggcagcggc tcccccaaga aaaaacgcaa ggtggaagat cctaagaaaa agcggaaagt 5700 ggacggcatt ggtagtggga gctaagcttc tctagctaga gtcgatcgac aagctcgagt 5760 ttctccataa taatgtgtga gtagttccca gataagggaa ttagggttcc tatagggttt 5820 cgctcatgtg ttgagcatat aagaaac cct tagtatgtat ttgtatttgt aaaatacttc 5880 tatcaataaa atttctaatt cctaaaacca aaatccagta ctaaaatcca gatc 5934 <210> 5 <211> 503 <212> PRT <213> Unknown <220> <223> Solanum lycopersicum <400> 5 Met Lys Ser Ser Leu Ser Ile Ser Phe Phe Lys Ile Met Met Ser Leu 1 5 10 15 Arg Val Lys Pro Phe Trp Ile Glu Leu Gly Tyr Phe Thr Thr Leu Ser 20 25 30 Leu Leu Gly Phe Leu Ala Leu Asn Tyr Val Ser Lys Pro Arg Thr Leu 35 40 45 Pro Ser Phe Arg Pro Gln Asn Leu Asp Val Leu Phe Thr Ser Val Ser 50 55 60 Ser Thr Thr Val Ser Ser Met Ser Thr Ile Glu Met Glu Val Phe Ser 65 70 75 80 Asn Val Gln Leu Val Phe Met Thr Ile Leu Met Phe Leu Gly Gly Glu 85 90 95 Ala Phe Thr Ser Phe Leu Ser Leu Lys Leu Ile Lys Asn Lys Glu Ser 100 105 110 Lys Asp Lys Ser Phe Ser Asn Lys Asp Tyr Glu Leu Gly Asn Val Ile 115 120 125 Asn Val Asp Asn Lys Leu Glu Asp Val Ile Ile Ile Asn Pro Ile Glu 130 135 140 Asp His Ile His Asp His His Asp Glu Ile Ile Lys Ile Lys Ser Ile 145 150 155 160 Lys Leu Leu Ser Asn Val Val Phe Gly Tyr Ile Leu Val Val Ile Leu 165 170 175 Leu Gly Ser Ser Leu Val Ser Leu Tyr Ile Ile Ile Ile Pro Ser Ala 180 185 190 Lys Gln Ile Leu Asp Gln Lys Gly Leu Asn Leu His Thr Phe Ser Leu 195 200 205 Phe Thr Thr Val Ser Thr Phe Ala Asn Cys Gly Phe Leu Pro Thr Asn 210 215 220 Glu Asn Met Met Ile Phe Lys Lys Asn Ser Gly Leu Leu Leu Ile Leu 225 230 235 240 Ile Pro Gln Val Leu Leu Gly Asn Thr Leu Phe Ala Pro Cys Leu Arg 245 250 255 Ile Val Ile Met Phe Leu Trp Lys Ile Thr Lys Arg His Glu Tyr Glu 260 265 270 Tyr Ile Leu Lys Asn Ser Lys Cys Val Gly Tyr Ser His Ile Phe Pro 275 280 285 Ser Tyr Glu Thr Ile Gly Ile Ala Ile Thr Val Val Val Gly Leu Ile Val 290 295 300 Phe Gln Phe Val Met Phe Cys Ser Leu Glu Trp Asn Ser Glu Gly Thr 305 310 315 320 Ser Gly Leu Ser Thr Tyr Glu Lys Ile Val Gly Ser Leu Phe Glu Val 325 330 335 Val Asn Thr Arg His Ala Gly Leu Ser Val Phe Asp Leu Ser Thr Phe 340 345 350 Thr Pro Ser Ile Leu Val Leu Phe Ala Leu Met Met Tyr Leu Ser Ser 355 360 365 Tyr Thr Thr Phe Leu Pro Val Asp Asn Tyr Glu Glu Lys Ser Glu Lys 370 375 380 Met Lys Lys Arg Lys Gly Arg Ser Leu Met Glu Tyr Ile Ser Leu Ser 385 390 395 400 Gln Pro Cys Cys Leu Val Ile Phe Thr Ile Leu Ile Cys Val Val Glu 405 410 415 Lys Asp Lys Met Lys Asn Asp Pro Leu Asn Phe Asn Val Leu Asn Ile 420 425 430 Leu Phe Glu Val Ile Ser Ala Tyr Gly Thr Val Gly Leu Ser Ile Gly 435 440 445 Ser Tyr Cys Ala Arg Gln Ile Asn Pro Asp Gly His Cys Lys Asp Val 450 455 460 Thr Tyr Gly Phe Ala Gly Lys Trp Ser Asn Thr Gly Lys Phe Ile Leu 465 470 475 480 Ile Ile Val Met Phe Phe Gly Arg Leu Lys Lys Tyr Asn Gln Arg Gly 485 490 495 Gly Lys Ala Trp Lys Val Leu 500

Claims (7)

Between the left border (LB) and right border (RB) sequences of the Ti plasmid, an antibiotic marker expression cassette; A donor sequence including a template sequence for homology-directed DNA repair (HDR) of HKT1;2 (High-affinity K+ transporter 1;2) gene derived from tomato ( Solanum lycopersicum ); dual HKT1;2 crRNA expression cassette; And an endonuclease (endonuclease) expression cassette; as a method for producing a genome-edited tomato plant with increased salt tolerance, comprising the step of transforming tomato plant cells with a recombinant vector comprising:
The template sequence for HDR of the tomato-derived HKT1;2 gene is a mutant in which the 217th asparagine (N) is substituted with aspartic acid (D) in the tomato HKT1;2 protein sequence consisting of the amino acid sequence of SEQ ID NO: 5. Characterized in that the coding sequence, a method for producing a genome-corrected tomato plant with increased salt tolerance. delete The method of claim 1, wherein the donor sequence consists of the nucleotide sequence of SEQ ID NO: 2. The method according to claim 1, wherein the antibiotic marker expression cassette, the crRNA expression cassette, and the endonuclease expression cassette are composed of the nucleotide sequences of SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 4, respectively. A genome-edited tomato plant with increased salt tolerance prepared by the method of any one of claims 1, 3 and 4. A seed whose genome has been corrected according to claim 5. Between the left border (LB) and right border (RB) sequences of the Ti plasmid, an antibiotic marker expression cassette; A donor sequence including a template sequence for homology-directed DNA repair (HDR) of a tomato-derived HKT1;2 (High-affinity K+ transporter 1;2) gene; dual HKT1;2 crRNA expression cassette; And an endonuclease (endonuclease) expression cassette; containing as an active ingredient a recombinant vector containing, as a genome editing composition for increasing the salt tolerance of tomato plants,
The template sequence for HDR of the tomato-derived HKT1;2 gene is a mutant in which the 217th asparagine (N) is substituted with aspartic acid (D) in the tomato HKT1;2 protein sequence consisting of the amino acid sequence of SEQ ID NO: 5. Characterized in that the coding sequence, a genome editing composition for increasing the salt tolerance of tomato plants.

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