CN109097387B - Method for creating purple tomato mutant by using CRISPR/Cas9 gene editing system and application - Google Patents

Abstract

The invention discloses a method for creating a purple tomato mutant by using a CRISPR/Cas9 gene editing system and application thereof, and relates to the technical field of tomato biotechnology and transgenosis. The invention constructs a CRISPR/Cas9-gRNA (pHSE401-gRNA) expression vector by designing a SlMYBATV mutation target site, introduces cultivated tomato LA1996 containing an Aft site by using an agrobacterium-mediated method, and screens by using a kanamycin resistance marker to obtain a positive transgenic plant; the tomato mutant which is determined by gene sequencing and phenotype observation and contains no foreign gene insertion and has homozygous mutation is a tomato mutant which completely loses the function of SlMYBATV and has purple fruits. The method can remarkably improve the content of anthocyanin in tomato peel, and has good application prospect in high-quality molecular breeding of tomatoes.

Description

Method for creating purple tomato mutant by using CRISPR/Cas9 gene editing system and application

Technical Field

The invention relates to the technical field of tomato biotechnology and transgenosis, in particular to a method for mutating tomato SlMYBATV genes and creating purple fruit tomato mutants by using a CRISPR/Cas9 gene editing system and application.

Background

The anthocyanin is a natural antioxidant substance widely existing in plants, and long-term eating of food rich in anthocyanin is beneficial to improving eyesight of people and reducing morbidity of cardiovascular diseases, obesity, diabetes, cancer and other diseases. The anthocyanin can enable the plant fruits to show rich colors (red, blue and purple), can meet the requirements of different consumers, and can improve the resistance of the plants to environmental stress, such as low temperature, drought, ultraviolet ray damage and the like.

Tomatoes are one of the most important vegetables in China and even in the world, and are also model crops in plant molecular biology research. Unlike other solanaceous vegetables, anthocyanins in cultivated tomatoes are mainly enriched in vegetative organs (stems and leaves), and fruits of the cultivated tomatoes hardly contain anthocyanins. The majority of the purple, brown (coffee) or black tomatoes found on the market are due to the co-existence of lycopene and chlorophyll in the ripe fruit. In wild tomato 3 sites have now been identified which are involved in the regulation of fruit Anthocyanin synthesis, Anthocynin fruit (Aft), Aubergine (Abg) and atroviolaceum (atv), respectively. The content of anthocyanin in tomato fruits can be obviously improved by introducing the Aft, Abg or atv locus into cultivated tomatoes by a genetic recombination method. However, a single genetic locus (Aft, Abg or atv) does not greatly increase the anthocyanin content in tomato fruits. For example, LA1996 tomato contains only ft sites and its fruit surface only appears purple spots. It was found that atv has an interaction with Aft and Abg respectively, while atv/atv Aft/Aft or atv/atv Abg/-tomato fruits containing two loci have significantly higher anthocyanin content than tomato fruits containing only one locus. atv, derived from wild tomato chessman Mannich (S. chessmaniae), is a recessive locus that is finely mapped to chromosome 7 within about 5.0 kb. The interval only contains one gene and codes R3 MYB transcription inhibitor, so the gene is named SlMYBATV. In the atv locus, a 4bp insertion exists in the coding region of the SlMYBATV gene, which causes frame shift mutation of the gene and premature termination of protein translation, so that the inhibition function of the gene on anthocyanin synthesis is invalid, and anthocyanin in the pericarp can be normally synthesized and accumulated to enable fruits to be purple.

CRISPR/Cas9(clustered regulated short palindromic repeats/CRISPR-associated nucleic acid 9, Cas9) gene editing technology is a new technology newly developed in recent years, and can realize site-directed mutation on a target gene without introducing external DNA. The CRISPR/Cas9 gene editing technology is used as a high-efficiency molecular operation technical means for plant genetic improvement and molecular breeding research, has low cost, simple operation and high mutation induction rate, is possible to avoid the current transgenic organism (GMO) regulation and has very wide application prospect. However, at present, no report is available for knocking out the SlMYBATV gene by using the technology to obtain the tomato with purple fruits.

Disclosure of Invention

In order to overcome the defects of long period, low efficiency and insufficient purple tomato resources in the traditional purple tomato breeding technology, the invention aims to provide a method for creating a purple tomato mutant by using a CRISPR/Cas9 gene editing system. The method uses CRISPR/Cas9 gene editing technology to perform site-directed mutagenesis on purple spot fruit tomato LA1996 to obtain purple fruit tomatoes which completely lose SlMYBATV functions, are stably inherited and have no foreign gene insertion.

The invention also aims to provide application of the solanum violaceum mutant prepared by the method for creating the solanum violaceum mutant by using the CRISPR/Cas9 gene editing system in tomato breeding.

The purpose of the invention is realized by the following technical scheme:

a method for creating a tomato mutant with purple fruits by using a CRISPR/Cas9 gene editing technology comprises the following steps:

(1) selection of gRNA target sites: according to literature reports, three transcripts (SlMYBATV-X1, SlMYBATV-X2 and SlMYBATV-X3) exist in SlMYBATV, and the first two exons of the three transcripts have no difference. Therefore, by using a CRISPR-Plant online design tool and according to the principle of designing target sites by using a CRISPR/cas9 technology, two target sites are designed on the second exon of the SlMYBATV gene, and the sequence distance between the two target sites is not more than 100 bp;

the sequence of the first gRNA target site is: 5'-gagtggttgcattagagac-3', respectively; (located 670 to 688 th from 5' end in SEQ ID NO: 1)

The sequence of the second gRNA target site is: 5'-acgaagaaacctctaaact-3', respectively; (from 694 to 712 positions 5' in SEQ ID NO: 1)

(2) Design of upstream and downstream primers of gRNA fragments: designing a gRNA PCR primer according to the principle of CRISPR/Cas9 primer design:

an upstream primer gRNA-F: 5'-ATATATGGTCTCGTTTGgtctctaatgcaaccactcGTTTTAGAGCTAGAAATAG-3', respectively;

a downstream primer gRNA-R: 5'-ATTATTGGTCTCGAAACacgaagaaacctctaaactCCAAACTACACTGTTAGATTC-3', respectively;

(3) construction of gRNA expression vector: carrying out PCR amplification and purification by using an upstream primer gRNA-F and a downstream primer gRNA-R by using a plasmid pCBC-DT1T2 as a template to obtain a gRNA fragment and purifying; the CRISPR/Cas9 vector plasmid and the purified gRNA fragment are respectively digested by endonuclease Bsa I; after purification, connecting the enzyme-digested CRISPR/Cas9 vector plasmid and the gRNA fragment by using T4 ligase to obtain a connection product; converting, screening and verifying the ligation product to obtain a CRISPR/Cas9-gRNA expression vector;

(4) introducing the CRISPR/Cas9-gRNA expression vector into agrobacterium to obtain CRISPR/Cas9-gRNA agrobacterium; wild tomato LA1996 (containing Aft locus) is used as a material for inducing callus, and the LA1996 tomato callus is infected by CRISPR/Cas9-gRNA agrobacterium;

(5) screening the calluses obtained in the step (4) for resistance to kanamycin, inducing to obtain regenerated seedlings, and screening to obtain transgenic positive plants;

(6) after a positive transgenic strain is obtained, extracting genome DNA, designing primers on two sides of a target site, carrying out PCR amplification on a target segment, carrying out sequencing after a PCR amplification product is purified, and judging whether the mutation is homozygous mutation or heterozygous mutation according to a sequencing result; if the mutation is homozygous, T is observed₀The color of the generation mutant tomato fruit is verified (homozygous mutation is purple), and T is collected₀Selfing seeds of mutant plants, sprouting, screening to obtain T₁Mutant with homozygous mutation of SlMYBATV and no foreign gene insertion in the generation plant. Recovering T if the sequencing result shows heterozygous mutation₀Selfing seeds of mutant plants, sprouting, screening to obtain T₁SlMYBATV homozygous mutant in generation plant and mutant without exo-gene insertion. The method for judging the presence or absence of foreign gene insertion comprises the following steps: designing primers according to foreign genes on CRISPR/Cas9 vector, carrying out PCR amplification on foreign target fragments, and judging T through 1.5% agarose gel electrophoresis₁Whether the generation mutant contains an insertion of a foreign gene.

(7) And (3) sequencing a gene target site, analyzing the insertion of the foreign gene and verifying the phenotype of the mutant to be a positive homozygous strain, namely the purple fruit tomato mutant which completely loses the function of SlMYBATV and has no insertion of the foreign gene.

The purple fruit tomato mutant prepared by the method for creating the purple fruit tomato mutant based on the CRISPR/Cas9 technology is applied to tomato breeding. Specifically, the purple fruit tomato mutant of SlMYBATV homozygous mutation without exorbitant gene insertion is selected as a material to carry out high-quality purple fruit tomato breeding through breeding technologies such as hybridization and the like.

In the step (1), the step (c),

the sequence of the SlMYBATV gene is shown as SEQ ID NO: 1 (4081 bp);

wherein the coding sequence (CDS) of SlMYBATV-X1 consists of SEQ ID NO: 1, 1-55 bases from 5' end, 661-774 bases, 868-950 bases, 2104-2370 bases.

The coding sequence (CDS) of SlMYBATV-X2 is expressed by SEQ ID NO: 1, 1-55 bases from 5' end, 661-774 bases, 868-950 bases, 3477-3494 bases.

The coding sequence (CDS) of SlMYBATV-X3 is expressed by SEQ ID NO: 1, 1-55 bases from 5' end, 661-774 bases, 868-953 bases.

Preferably, the CRISPR/Cas9 vector described in step (3) is pHSE 401.

The PCR amplification system in the step (3) is as follows: pHSE401 plasmid 1 μ L, KAPA HiFi HotStart ReadyMix 25 μ L, gRNA-F2 μ L, gRNA-R2 μ L, and ddH₂O to 50 μ L; the PCR amplification procedure was: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 1min, and 32 cycles; extending for 5min at 72 ℃;

the gRNA fragment enzyme digestion system in the step (3) is as follows: gRNA fragment 2. mu.g, 10 × CutSmart Buffer 5. mu.L, Bsa I10U, plus ddH₂O to 50 μ L; the enzyme cutting system of the CRISPR/Cas9 carrier plasmid is as follows: CRISPR/Cas9 vector plasmid 2. mu.g, 10 × CutSmart Buffer 5. mu.L, BsA 10U, plus ddH₂O to 50 μ L;

the enzyme cutting conditions in the step (3) are as follows: carrying out enzyme digestion for 2h at 37 ℃;

the T4 ligase linker system in the step (3) is as follows: enzyme-digested and purified 8 mu L of CRISPR/Cas9 vector plasmid, enzyme-digested and purified 4 mu L of gRNA fragment, 5 mu L of T4 DNA ligase buffer solution (10X), 1 mu L of T4 DNA ligase and ddH₂O to 50 μ L;

the T4 ligase connection conditions in the step (3) are as follows: ligation was carried out at 16 ℃ for 8 h.

The PCR product or the enzyme digestion product in the step (3) is purified by using a Tiangen Universal DNA purification and recovery kit (DP 214).

Preferably, the agrobacterium in step (4) is agrobacterium tumefaciens AGL 1.

Compared with the prior art, the invention has the following advantages and effects:

the invention constructs a CRISPR/Cas9-gRNA (pHSE401-gRNA) expression vector by designing a SlMYBATV mutation target site, introduces cultivated tomato LA1996 containing an Aft site by using an agrobacterium-mediated method, and screens by using a kanamycin resistance marker to obtain a positive transgenic plant; the tomato mutant which is determined by gene sequencing and phenotype observation and contains no foreign gene insertion and has homozygous mutation is a tomato mutant which completely loses the function of SlMYBATV and has purple fruits. The method can remarkably improve the content of anthocyanin in tomato peel, and has good application prospect in high-quality molecular breeding of tomatoes.

Drawings

FIG. 1 is a construction of a gRNA expression vector (pHSE 401-gRNA); wherein, A: PCR product electrophoresis chart of gRNA fragment; b: connecting a gRNA fragment with an expression vector to obtain a position sequencing map; c: pHSE401-gRNA schematic.

FIG. 2 is partial exon sequences of Wild Type (WT) and mutant (M1, M2 and M3) genes of SlMYBATV; wherein, A: a partial exon sequence variation diagram of SlMYBATV gene WT and M1, M2 and M3 mutant strains; b: sequencing maps of target site mutations of SlMYBATV genes WT and M1, M2 and M3 mutant strains.

FIG. 3 is a phenotypic analysis of wild type plants (WT) and mutant lines (M1) of SlMYBATV; wherein, A: leaf blades of a SlMYBATV wild-type plant (WT) and a mutant strain (M1); b: fruits of a SlMYBATV wild-type plant (WT) and mutant strain (M1); c: anthocyanin content in leaves and fruit pericarp of SlMYBATV wild type plants (WT) and mutant lines (M1).

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

The Tomato variety used in the present invention is wild type Tomato LA1996, derived from Tomato Genetics Resource Center (TGRC).

Plasmids pCBC-DT1T2 and pHSE401 are disclosed in the documents "A CRISPR/Cas9 toolkit for multiplex genome editing in plants", Bmc Plant Biology, 2014,14(1):327 ". Plasmid pCBC-DT1T2, plasmid pHSE401 are both available from Addgene (http:// www.addge ne. org /).

The first embodiment is as follows:

1. selection of target site sequence of SlMYBATV gene and design of upstream and downstream primers of gRNA fragment

According to literature reports, three transcripts (SlMYBATV-X1, SlMYBATV-X2 and SlMYBATV-X3) exist in SlMYBATV, and the first two exons of the three transcripts have no difference. Therefore, by using a CRISPR-Plant online design tool and according to the principle of designing target sites by using a CRISPR/cas9 technology, two target sites are designed on the second exon of the SlMYBATV gene (see SEQ ID NO: 1), and the sequence distance between the two target sites is not more than 100 bp. The sequence of the first gRNA target site of SlMYBATV is: 5'-gagtggttgcattagagac-3', respectively; the sequence of the second gRNA target site is: 5'-acgaagaaacctctaaact-3' are provided.

2. Designing gRNA PCR primers according to the sequence of gRNA target sites:

an upstream primer gRNA-F: 5'-ATATATGGTCTCGTTTGgtctctaatgcaaccactcGTTTTAGAGCTAGAAATAG-3', respectively;

a downstream primer gRNA-R: 5'-ATTATTGGTCTCGAAACacgaagaaacctctaaactCCAAACTACACTGTTAGATTC-3', respectively;

3. construction of gRNA expression vector:

3.1 using pCBC-DT1T2 as a template, using upstream primer gRNA-F and downstream primer gRNA-R to perform PCR amplification and purification, to obtain gRNA fragment (FIG. 1A). The PCR amplification system is as follows:

pCBC-DT1T2	1μL
		KAPA HiFi HotStart ReadyMix(2×)(KAPA,cat.no.KK2601)	25μL
gRNA-F	2μL
		gRNA-R	2μL
ddH2O	20μL

the PCR amplification procedure was: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 1min, and 32 cycles; extension at 72 ℃ for 5 min. PCR product purification a tiangen Universal DNA purification recovery kit (tiangen, cat No. dp214) was used.

3.2 the pHSE401 plasmid and the purified gRNA fragment were digested separately with the endonuclease Bsa I (NEB, cat. No. R353535V). The enzyme digestion reaction system is as follows:

pHSE401 plasmid/gRNA fragment	2μg
		10×CutSmart Buffer	5μL
Bsa Ⅰ	10U
		ddH2O	Adding to 50 μ L

And carrying out enzyme digestion reaction at 37 ℃ for 2h, and then purifying the pHSE401 plasmid and the gRNA fragment after enzyme digestion by using a Tiangen Universal DNA purification recovery kit.

3.3 ligation of the purified linear pHSE401 plasmid and gRNA fragment with T4 DNA ligase (NEB, cat. No. M0202V) gave a ligation product. Connecting a reaction system:

linear pHSE401 plasmid	1μL
		gRNA fragments	25μL
T4 DNA ligase buffer (10X)	2μL
		T4 DNA ligase	2μL
ddH2O	Adding to 50 μ L

4. Transforming the ligated product into escherichia coli competent DH5 α (Takara, cat No.9057), plating overnight, picking out a single colony, shaking for 6h, extracting a plasmid by using a plasmid extraction kit (bio, cat No. dp105), and then performing sample sequencing, wherein the sequencing sequence is scprimer 1: 5'-ACGACGGCCAGTGCCAAG-3' are provided. The results of the sequencing part are shown in FIG. 1B.

5. Transforming Agrobacterium tumefaciens AGL1 (conventionally commercially available) with the correctly ligated pHSE401-gRNA plasmid (FIG. 1C) to obtain pHSE401-gRNA Agrobacterium; wild tomato LA1996 (containing Aft locus) is used as a material for inducing callus, agrobacterium is used for mediating and transforming LA1996 tomato callus, kanamycin resistance screening is carried out, and 3 transgenic positive strains are obtained by differentiation and regeneration of the resistant callus and are marked as M1, M2 and M3.

6. Detection of SlMYBATV gene mutant in transgenic tomato

6.1 designing a target gene detection primer, respectively designing primers at the upstream and downstream of two gRNA sequences according to a target gene, wherein the primer sequences are respectively as follows:

SlMYBATV-F：5'-TATTACACTACTTATAAGTTCACAATTAA-3'；

SlMYBATV-R：5'-AAAAATATTAAACGTACCTCTCTCTA-3'；

6.2 the genomic DNA (Tiangen, cat No. DP305) of the 3 transgenic positive plants and the control plants (wild type tomato LA1996, WT) were extracted separately. PCR was carried out using the above DNA as a template. And (3) PCR reaction system:

positive plant DNA/control group plant DNA	1μL
		KAPA HiFi HotStart ReadyMix(2×)	25μL
SlMYBATV-F	2μL
		SlMYBATV-R	2μL
ddH2O	20μL

The PCR amplification procedure was: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s, for 32 cycles; extension at 72 ℃ for 5 min. And purifying the PCR product by using the Tiangen Universal DNA purification recovery kit, and sequencing, wherein a sequencing company is a Shanghai organism, and the sequence of a sequencing primer is SlMYBATV-F.

6.3 judging whether the mutation is homozygous mutation or heterozygous mutation according to the sequencing result. If the mutation is homozygous, T is observed₀The color of the generation mutant tomato fruit is verified (homozygous mutation is purple), and T is collected₀Selfing seeds of mutant plants, sprouting, screening to obtain T₁Mutant with homozygous mutation of SlMYBATV and no foreign gene insertion in the generation plant. Recovering T if the sequencing result shows heterozygous mutation₀Selfing seeds of mutant plants, sprouting, screening to obtain T₁The mutant of SlMYBATV homozygous mutant in the generation plant and without exo-gene insertion, and the screening method is the same as above. The method for judging the presence or absence of foreign gene insertion comprises the following steps: designing a primer according to the pHSE401 vector sequence, wherein the primer sequence is as follows:

NPTⅡ-F：5'-TTGTCACTGAAGCGGGAAG-3'；

NPTⅡ-R：5'-CCGTAAAGCACGAGGAAGC-3'；

PCR amplification is carried out on the transgenic plant, and T is judged by 1.5 percent agarose gel electrophoresis₁Whether the generation mutant contains an insertion of a foreign gene.

According to the analysis of the sequencing results, it was found that there was a deletion of 1bp in M1 strain, a deletion of a small fragment of 2bp in M2 strain and a deletion of a small fragment of 7bp in M3 strain (FIG. 2). Planting M1, M2 and M3 strains and wild type tomato LA1996 strains in a plastic greenhouse (Guangzhou), wherein the anthocyanin content of all transgenic positive plants, namely tomato leaves, has no obvious difference from wild type tomatoes in the green mature period and the red mature period of fruits; the tomato fruits of all transgenic positive plant lines present purple black, while the wild type tomato fruits only present a few purple spots, and the anthocyanin content analysis shows that the anthocyanin content in all transgenic positive plant fruits is remarkably higher than that in the wild type tomato LA1996, and the results are shown in Table 1 and figure 3. Among them, the method for measuring the anthocyanin content is referred to in the "transport analysis in high-affinity in microorganisms synthesized effective of air and atv genes" Journal of Plant Physiology ", 2011,168(3): 270-.

TABLE 1 content of anthocyanins in wild type tomato and SlMYBATV mutant tomato (mg/100g FW)

Location of a body part	WT	M1	M2	M3
					Blade	2.85±0.34	2.45±0.32	2.93±0.29	3.04±0.35
Epicarp (Green ripe period)	13.00±1.98	314.42±24.38	348.259±29.98	286.69±20.25
					Epicarp (red ripe period)	11.00±1.76	244.42±38.32	308.259±28.74	266.69±25.32

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

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<211> 55

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> upstream primer gRNA-F

<400> 4

atatatggtc tcgtttggtc tctaatgcaa ccactcgttt tagagctaga aatag 55

<210> 5

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> downstream primer gRNA-R

<400> 5

attattggtc tcgaaacacg aagaaacctc taaactccaa actacactgt tagattc 57

<210> 6

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> scprimer1

<400> 6

acgacggcca gtgccaag 18

<210> 7

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> SlMYBATV-F

<400> 7

tattacacta cttataagtt cacaattaa 29

<210> 8

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> SlMYBATV-R

<400> 8

aaaaatatta aacgtacctc tctcta 26

<210> 9

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> NPTⅡ-F

<400> 9

ttgtcactga agcgggaag 19

<210> 10

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> NPTⅡ-R

<400> 10

ccgtaaagca cgaggaagc 19

Claims (7)

1. A method for creating a tomato mutant with purple fruits by using a CRISPR/Cas9 gene editing technology is characterized by comprising the following steps:

including a gRNA target designed inSlMYBATVOn the second exon of the gene, and the distance between the sequences of two target sites is not more than 100 bp;

the sequence of the first gRNA target site is: 5'-gagtggttgcattagagac-3', respectively;

the sequence of the second gRNA target site is: 5'-acgaagaaacctctaaact-3', respectively;

including gRNA PCR primers:

an upstream primer gRNA-F: 5'-ATATATGGTCTCGTTTGgtctctaatgcaaccactcGTTTTAGAGCTAGAAATAG-3', respectively;

a downstream primer gRNA-R: 5'-ATTATTGGTCTCGAAACacgaagaaacctctaaactCCAAACTACACTGTTAGATTC-3', respectively;

the method comprises the following steps:

(1) selection of gRNA target sites: designing both target sites atSlMYBATVOn the second exon of the gene, and the distance between the sequences of two target sites is not more than 100 bp;

the sequence of the first gRNA target site is: 5'-gagtggttgcattagagac-3', respectively;

the sequence of the second gRNA target site is: 5'-acgaagaaacctctaaact-3', respectively;

(2) design of gRNA PCR primers:

an upstream primer gRNA-F: 5'-ATATATGGTCTCGTTTGgtctctaatgcaaccactcGTTTTAGAGCTAGAAATAG-3', respectively;

a downstream primer gRNA-R: 5'-ATTATTGGTCTCGAAACacgaagaaacctctaaactCCAAACTACACTGTTAGATTC-3', respectively;

(3) construction of gRNA expression vector:

carrying out PCR amplification and purification by using an upstream primer gRNA-F and a downstream primer gRNA-R by using a plasmid pCBC-DT1T2 as a template to obtain a gRNA fragment and purifying; using endonucleasesBsaI, respectively carrying out enzyme digestion on a CRISPR/Cas9 vector plasmid and the purified gRNA fragment; after purificationConnecting the enzyme-cut CRISPR/Cas9 vector plasmid and the gRNA fragment by using T4 ligase to obtain a connection product; converting, screening and verifying the ligation product to obtain a CRISPR/Cas9-gRNA expression vector;

the CRISPR/Cas9 vector is pHSE 401;

(4) introducing the CRISPR/Cas9-gRNA expression vector into agrobacterium to obtain CRISPR/Cas9-gRNA agrobacterium; infection of Agrobacterium with CRISPR/Cas9-gRNA containingAftSite wild type tomato LA1996 callus; the agrobacterium is agrobacterium tumefaciens AGL 1;

(5) screening the calluses obtained in the step (4) for resistance to kanamycin, inducing to obtain regenerated seedlings, and screening to obtain transgenic positive plants;

(6) obtaining positive transgenic line, sequencing gene target site, inserting foreign gene, analyzing mutant phenotype to obtain positive homozygous line, i.e. completely lost homozygous lineSlMYBATVTomato mutants of the purple fruit that are functional and have no foreign gene insertion.

2. The method for creating the tomato mutant with purple fruit by using CRISPR/Cas9 gene editing technology as claimed in claim 1, wherein the method comprises the following steps:

the PCR amplification system in the step (3) is as follows: pHSE401 plasmid 1 μ L, KAPA HiFi HotStart ReadyMix 25 μ L, gRNA-F2 μ L, gRNA-R2 μ L, and ddH₂O to 50 μ L; the PCR amplification procedure was: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 1min, and 32 cycles; extension at 72 ℃ for 5 min.

3. The method for creating the tomato mutant with purple fruit by using CRISPR/Cas9 gene editing technology as claimed in claim 1, wherein the method comprises the following steps:

the gRNA fragment enzyme digestion system in the step (3) is as follows: 2 ug of gRNA fragment, 5 ul of 10 × CutSmart Buffer,BsaI10U, plus ddH₂O to 50 μ L; the enzyme cutting system of the CRISPR/Cas9 carrier plasmid is as follows: 2 mu g of CRISPR/Cas9 carrier plasmid, 5 mu L of 10 multiplied by CutSmart Buffer,BsaI10U, plus ddH₂O to 50. mu.L.

4. The method for creating the tomato mutant with purple fruit by using CRISPR/Cas9 gene editing technology as claimed in claim 1, wherein the method comprises the following steps:

the enzyme cutting conditions in the step (3) are as follows: the enzyme was cleaved at 37 ℃ for 2 h.

5. The method for creating the tomato mutant with purple fruit by using CRISPR/Cas9 gene editing technology as claimed in claim 1, wherein the method comprises the following steps:

the T4 ligase linker system in the step (3) is as follows: enzyme-digested and purified 8 mu L of CRISPR/Cas9 vector plasmid, enzyme-digested and purified 4 mu L of gRNA fragment, 5 mu L of 10 XT 4 DNA ligase buffer solution, 1 mu L of T4 DNA ligase, and ddH₂O to 50. mu.L.

6. The method for creating the tomato mutant with purple fruit by using CRISPR/Cas9 gene editing technology as claimed in claim 1, wherein the method comprises the following steps:

the T4 ligase connection conditions in the step (3) are as follows: ligation was carried out at 16 ℃ for 8 h.

7. The method for creating the tomato mutant with purple fruits by using CRISPR/Cas9 gene editing technology as claimed in any one of claims 1-6, and the application of the method in tomato breeding.

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