CN109097387A

CN109097387A - A kind of methods and applications with CRISPR/Cas9 gene editing system initiative purple fruit Tomato mutants

Info

Publication number: CN109097387A
Application number: CN201810895852.XA
Authority: CN
Inventors: 邱正坤; 曹必好; 侯军晓; 黄泽军; 崔霞
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2018-08-08
Filing date: 2018-08-08
Publication date: 2018-12-28
Anticipated expiration: 2038-08-08
Also published as: CN109097387B

Abstract

The present invention discloses a kind of methods and applications with CRISPR/Cas9 gene editing system initiative purple fruit Tomato mutants, is related to tomato biological technology and field of transgenic technology.The present invention is mutated target site by design SlMYBATV, construct CRISPR/Cas9-gRNA (pHSE401-gRNA) expression vector, using agrobacterium-mediated transformation import the cultivated tomato LA1996 containing the site Aft, with card receive chloramphenicol resistance label screening acquisition positive transgenic plant；The homozygous mutation determined through gene sequencing and Phenotypic Observation and the strain without the insertion of foreign aid's gene, as completely lose SlMYBATV function, fruit Tomato mutants purple.The present invention can it is extremely significant improve tomato peel in anthocyanidin content, have a good application prospect in tomato high-quality molecular breeding.

Description

It is a kind of to formulate purple fruit Tomato mutants with CRISPR/Cas9 gene editing system Methods and applications

Technical field

The present invention relates to tomato biological technology and field of transgenic technology, and in particular to uses CRISPR/Cas9 to a kind of Gene editing system causes the methods and applications of mutation, initiative purple fruit Tomato mutants to tomato SlMYBATV gene.

Background technique

Anthocyanidin is to be widely present a kind of natural antioxidants, long-term consumption in plant to be rich in the food of anthocyanidin Object, which is conducive to people, improves eyesight, reduces the disease incidence of the diseases such as cardiovascular disease, obesity, diabetes and cancer.Anthocyanidin can So that color abundant (red, blue and purple) is presented in fruit, different consumer demands, while cyanine can satisfy Element can also improve plant to the resistivity of environment-stress, such as low temperature, arid and uv damage.

Tomato be it is Chinese so that in the world one of most important vegetables and molecular biology of plants research in mode make Object.Different from other solanaceous vegetables, anthocyanidin is mainly enriched in nutrition organs (stem, leaf) in cultivated tomato, several in fruit Anthocyanidin is not contained.Seen most of purple, brown (coffee color) or black tomato are due to ripening fruits in the market Middle lycopene and chlorophyll and caused by depositing.3 participation fruit anthocyanidin synthesis have been identified in wild-type tomato to adjust The site of control, respectively Anthocyanin fruit (Aft), Aubergine (Abg) and atroviolacium (atv).Pass through The site Aft, Abg or atv imported into cultivated tomato and is remarkably improved anthocyanidin in tamato fruit by the method for Genetic Recombination Content.However, single genetic locus (Aft, Abg or atv) cannot greatly improve the content of anthocyanidin in tamato fruit.For example, LA1996 tomato only contains the site Aft, and purple dot is only presented in fruit surface.The study found that atv is deposited with Aft and Abg respectively In interaction, while atv/atv Aft/Aft or atv/atv Abg/- contain the tamato fruit anthocyanidin content there are two site It is significantly higher than the tamato fruit containing only a site.The site atv, this Manny tomato of source wild-type tomato contract It (S.cheesmaniae), is a recessive site, by within the scope of No. 7 chromosome about 5.0kb of finely positioning.The section contains only There is a gene, encodes R3 MYB transcription inhibitory factor, therefore the gene is named as SlMYBATV.In the site atv, The gene coding region SlMYBATV results in the gene frameshift mutation and protein translation terminates in advance, make there are the insertion of a 4bp Its inhibition disabler to anthocyanidin synthesis, anthocyanidin normally can be synthesized and be accumulated and make fruit that purple be presented in pericarp.

CRISPR/Cas9(clustered regularly interspaced short palindromic Repeats/CRISPR-associated nuclease 9, Cas9) gene editing technology be new development in recent years get up one The new technology of kind, it can realize rite-directed mutagenesis to target gene in the case where not introducing foreign aid DNA.CRISPR/Cas9 base Molecule manipulation technological means because of editing technique as efficient a genetic modification of plants and molecular breeding research, cost Low, easy to operate, mutation inductivity height, and be possible to evade current genetically modified organism (GMO) regulation, application prospect is very wide It is wealthy.But the report that there is no knock out SlMYBATV gene with the technology at present, obtain purple fruit tomato.

Summary of the invention

In order to overcome the shortcomings of asking for long traditional purple fruit tomato breeding technology cycle, low efficiency and purple fruit tomato resources scarcity Topic, the purpose of the present invention is to provide a kind of sides with CRISPR/Cas9 gene editing system initiative purple fruit Tomato mutants Method.This method uses CRISPR/Cas9 gene editing technology rite-directed mutagenesis purple dot fruit tomato LA1996, complete to obtain It loses SlMYBATV function, stablize heredity and the purple fruit tomato without the insertion of foreign aid's gene.

Another object of the present invention is to provide formulate purple fruit kind with CRISPR/Cas9 gene editing system by above-mentioned Application of the purple fruit Tomato mutants of the method preparation of eggplant mutant in tomato breeding.

The purpose of the invention is achieved by the following technical solution:

A method of purple fruit Tomato mutants being formulated with CRISPR/Cas9 gene editing technology, including following Step:

(1) selection of gRNA target site: according to the literature, SlMYBATV there are three kinds of transcripts (SlMYBATV-X1, SlMYBATV-X2 and SlMYBATV-X3), the first two exon of three transcripts does not have difference.Therefore, CRISPR- is utilized Plant Photographing On-line tool designs two target sites according to the principle of CRISPR/cas9 Technology design target site On second exon of SlMYBATV gene, and two target site sequence spacing are no more than 100bp；

The sequence of first gRNA target site are as follows: 5'-gagtggttgcattagagac-3'；(it is located in SEQ ID NO:1 From 5 ' the 670th~688, ends)

The sequence of second gRNA target site are as follows: 5'-acgaagaaacctctaaact-3'；(it is located in SEQ ID NO:1 From 5 ' the 694th~712, ends)

(2) design of gRNA segment upstream and downstream primer: according to the principle of CRISPR/Cas9 design of primers, gRNA is designed PCR primer:

Upstream primer gRNA-F:5'-ATATATGGTCTCGTTTGgtctctaatgcaaccactcGTTTTAGAGC TAGA AATAG-3'；

Downstream primer gRNA-R:5'-ATTATTGGTCTCGAAACacgaagaaacctctaaactCCAAACTACA CTGT TAGATTC-3'；

(3) building of gRNA expression vector: using plasmid pCBC-DT1T2 as template, drawn with upstream primer gRNA-F and downstream Object gRNA-R carries out PCR amplification, purifying, obtains gRNA segment and purifies；Distinguish digestion CRISPR/Cas9 with restriction endonuclease Bsa I to carry Constitution grain and the gRNA segment after purification；CRISPR/Cas9 carrier after connecting the digestion with T4 ligase after purification Plasmid and gRNA segment, obtain connection product；By connection product conversion, screening, verify to get CRISPR/Cas9-gRNA expression Carrier；

(4) CRISPR/Cas9-gRNA expression vector is imported in Agrobacterium, obtains CRISPR/Cas9-gRNA Agrobacterium； With wild-type tomatoes LA1996 (containing the site Aft) for material evoked callus, invaded with CRISPR/Cas9-gRNA Agrobacterium Contaminate LA1996 Tomato Calli；

(5) callus for obtaining step (4) receives chloramphenicol resistance screening through card, induces and obtain after regrowth through screening Obtain transgenic positive plant；

(6) after obtaining positive transgenic strain, genomic DNA is extracted, in the two sides design primer of target site, to purpose piece Duan Jinhang PCR amplification, purify pcr amplification product after be sequenced, according to sequencing result judge be mutated whether be homozygous mutation or Heterozygous mutant；If sporting homozygous mutation, T is observed₀Verified that (homozygous mutation is purple for mutant Tomato Fruit Color Color), collect T₀For mutant plants selfed seed, seedling is sent out, screening obtains T₁For SlMYBATV homozygous mutation in plant and without outer Help the mutant of gene insertion.If sequencing result is shown as heterozygous mutant, T is collected₀For mutant plants selfed seed, hair Seedling, screening obtain T₁For SlMYBATV Mutants homozygous in plant and the mutant without the insertion of foreign aid's gene.Determine whether outer The method for helping gene insertion: according to foreign aid's gene design primer on CRISPR/Cas9 carrier, PCR is carried out to foreign aid's target fragment Amplification, judges T through 1.5% agarose gel electrophoresis₁Whether contain the insertion of foreign aid's gene for mutant.

(7) positive homozygous lines are verified as through the sequencing of gene target site, foreign aid's gene insertion analysis and mutation type surface, As completely lose SlMYBATV function and the purple fruit Tomato mutants without the insertion of foreign aid's gene.

By the purple fruit kind of the above-mentioned method preparation based on CRISPR/Cas9 technology initiative purple fruit Tomato mutants Application of the eggplant mutant in tomato breeding.Specifically, selecting the SlMYBATV homozygous mutation and without the insertion of foreign aid's gene Purple fruit Tomato mutants are that material passes through the breeding techniques such as hybridization progress high-quality purple fruit tomato breeding.

In step (1),

The SlMYBATV gene order is as shown in SEQ ID NO:1 (4081bp)；

Wherein, the coded sequence (CDS) of SlMYBATV-X1 by SEQ ID NO:1 from 5 ' end the 1st~55 bit bases, 661st~774 bit bases, the 868th~950 bit bases, the 2104th~2370 bit bases composition.

The coded sequence (CDS) of the SlMYBATV-X2 by SEQ ID NO:1 from 5 ' end the 1st~55 bit bases, 661st~774 bit bases, the 868th~950 bit bases, the 3477th~3494 bit bases composition.

The coded sequence (CDS) of the SlMYBATV-X3 by SEQ ID NO:1 from 5 ' end the 1st~55 bit bases, 661st~774 bit bases, the 868th~953 bit bases.

Preferably, CRISPR/Cas9 carrier described in step (3) is pHSE401.

PCR amplification system described in step (3) are as follows: 1 μ L, KAPA HiFi HotStart of pHSE401 plasmid 25 22 μ L of μ L, gRNA-R of μ L, gRNA-F of ReadyMix, adds ddH₂O to 50 μ L；PCR amplification program are as follows: 95 DEG C of initial denaturations 3min；95 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 1min, 32 recycle；72 DEG C of extension 5min；

GRNA segment digestion system described in step (3) are as follows: 2 μ g, 10 × CutSmart Buffer of gRNA segment, 5 μ I 10U of L, Bsa, adds ddH₂O to 50 μ L；CRISPR/Cas9 vector plasmid digestion system are as follows: 2 μ g of CRISPR/Cas9 vector plasmid, 10 × CutSmart Buffer, 5 I 10U of μ L, Bsa, adds ddH₂O to 50 μ L；

Digestion condition described in step (3) are as follows: 37 DEG C of digestion 2h；

T4 ligase linked system described in step (3) are as follows: digestion, 8 μ of CRISPR/Cas9 vector plasmid after purification L, digestion, 4 μ L, T4 DNA ligase buffer (10 ×) of gRNA segment, 5 μ L, T4 DNA ligase, 1 μ L after purification, adds ddH₂O to 50 μ L；

The condition of the connection of T4 ligase described in step (3) are as follows: 16 DEG C of connection 8h.

PCR product described in step (3) or digestion products purifying use Tiangeng Universal DNA purification and recovery Kit (DP214).

Preferably, Agrobacterium described in step (4) is Agrobacterium tumefaciems AGL1.

The present invention has the following advantages and effects with respect to the prior art:

The present invention is mutated target site by design SlMYBATV, constructs CRISPR/Cas9-gRNA (pHSE401-gRNA) table Up to carrier, the cultivated tomato LA1996 containing the site Aft is imported using agrobacterium-mediated transformation, chloramphenicol resistance label screening is received with card Obtain positive transgenic plant；The homozygous mutation determined through gene sequencing and Phenotypic Observation and the strain without the insertion of foreign aid's gene System, as completely loses SlMYBATV function, fruit Tomato mutants purple.The present invention can extremely significant raising tomato fruit The content of anthocyanidin in skin has a good application prospect in tomato high-quality molecular breeding.

Detailed description of the invention

Fig. 1 is the building of gRNA expression vector (pHSE401-gRNA)；Wherein, A:gRNA fragment PCR products electrophoretogram；B: GRNA segment and expression vector link position Sequencing chromatogram；C:pHSE401-gRNA schematic diagram.

Fig. 2 is SlMYBATV gene wild type (WT) and mutant (M1, M2 and M3) partial exon sequences；Wherein, A: SlMYBATV gene WT and M1, M2, M3 mutating strain series partial exon sequences variation figure；B:SlMYBATV gene WT and M1, M2, M3 mutating strain series target site Mutational part Sequencing chromatogram.

Fig. 3 is SlMYBATV WT lines (WT) and mutant strain (M1) phenotypic analysis；Wherein, A:SlMYBATV is wild Raw type plant (WT) and mutant strain (M1) blade；B:SlMYBATV WT lines (WT) and mutant strain (M1) fruit It is real；Anthocyanidin content in C:SlMYBATV WT lines (WT) and mutant strain (M1) blade and fruit rind.

Specific embodiment

Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited In this.

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

Plasmid pCBC-DT1T2, plasmid pHSE401 are in document " A CRISPR/Cas9 toolkit for Multiplex genome editing in plants. " Bmc Plant Biology ", 2014,14 (1): public in 327. " It opens.Plasmid pCBC-DT1T2, plasmid pHSE401 are purchased from Addgene (http://www.addge ne.org/).

Embodiment one:

1, the design of the selection of SlMYBATV gene target site sequence and gRNA segment upstream and downstream primer

According to the literature, SlMYBATV there are three kinds of transcripts (SlMYBATV-X1, SlMYBATV-X2 and SlMYBATV-X3), the first two exon of three transcripts does not have difference.Therefore, CRISPR-Plant Photographing On-line work is utilized Tool, according to the principle of CRISPR/cas9 Technology design target site, designs two target sites the second of SlMYBATV gene (see SEQ ID NO:1) on a exon, and two target site sequence spacing are no more than 100bp.First gRNA of SlMYBATV The sequence of target site are as follows: 5'-gagtggttgcattagagac-3'；The sequence of second gRNA target site are as follows: 5'- acgaagaaacctctaaact-3'。

2, according to the sequence of gRNA target site, gRNA PCR primer is designed:

3, the building of gRNA expression vector:

3.1 using plasmid pCBC-DT1T2 as template, carries out PCR expansion with upstream primer gRNA-F and downstream primer gRNA-R Increase, purifying, obtains gRNA segment (Figure 1A).PCR amplification system are as follows:

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

PCR amplification program are as follows: 95 DEG C of initial denaturation 3min；95 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 1min, 32 A circulation；72 DEG C of extension 5min.PCR product purifying using Tiangeng Universal DNA purification and recovery kit (Tiangeng, cat.no.DP214)。

3.2 with restriction endonuclease Bsa I (NEB, cat.no.R3535V) respectively digestion pHSE401 plasmids and it is described after purification GRNA segment.Endonuclease reaction system are as follows:

PHSE401 plasmid/gRNA segment	2μg
		10×CutSmart Buffer	5μL
Bsa Ⅰ	10U
		ddH₂O	Add to 50 μ L

37 DEG C of endonuclease reaction 2h, after then using Tiangeng Universal DNA purification and recovery kit respectively to digestion PHSE401 plasmid and gRNA fragment purification.

3.3 use T4 DNA ligase (NEB, cat.no.M0202V) by linear pHSE401 plasmid and gRNA piece after purification Section connection, obtains connection product.Coupled reaction system:

Linear pHSE401 plasmid	1μL
		GRNA segment	25μL
T4 DNA ligase buffer (10 ×)	2μL
		T4 DNA ligase	2μL
ddH₂O	Add to 50 μ L

4, the product after connection is converted into E. coli competent DH5 α (Takara, cat.no.9057), plated overnight, Picking single colonie shakes bacterium 6h, carries out sample survey after extracting plasmid using plasmid extraction kit (raw work, cat.no.DP105) Sequence, sequencing sequence scprimer1:5'-ACGACGGCCAGTGCCAAG-3'.It is as shown in Figure 1B that partial results are sequenced.

5, correct pHSE401-gRNA plasmid (Fig. 1 C) conversion Agrobacterium tumefaciems AGL1 (conventional commercial) will be connected, will be obtained PHSE401-gRNA Agrobacterium；With wild-type tomatoes LA1996 (containing the site Aft) for material evoked callus, with Agrobacterium Mediated transformation LA1996 Tomato Calli, through card receive chloramphenicol resistance screening, resistant calli differentiation and regeneration obtain transgenosis Positive strain 3, is denoted as M1, M2, M3.

6, in transgene tomato SlMYBATV gene mutation body detection

6.1 purpose of design genetic test primers are separately designed according to target gene in two gRNA Sequences upstreams and downstream Primer, primer sequence are respectively as follows:

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

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

6.2 extract 3 transgenic positive plant and control group plant obtained (wild-type tomatoes LA1996, note respectively For WT) genomic DNA (Tiangeng, cat.no.DP305).PCR reaction is carried out by template of above-mentioned DNA.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

PCR amplification program are as follows: 95 DEG C of initial denaturation 3min；95 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 30s, 32 Circulation；72 DEG C of extension 5min.It using Tiangeng Universal DNA purification and recovery kits PCR product, is sequenced, is surveyed Sequence company is the raw work biology in Shanghai, and sequencing primer sequence is above-mentioned SlMYBATV-F.

6.3 sport homozygous mutation or heterozygous mutant according to sequencing result judgement.If sporting homozygous mutation, observation T₀(homozygous mutation is purple) is verified for mutant Tomato Fruit Color, collects T₀For mutant plants selfed seed, hair Seedling, screening obtain T₁For SlMYBATV homozygous mutation in plant and the mutant without the insertion of foreign aid's gene.If sequencing result It is shown as heterozygous mutant, collects T₀For mutant plants selfed seed, seedling is sent out, screening obtains T₁It is homozygous for SlMYBATV in plant Mutant and the mutant for being free of the insertion of foreign aid's gene, screening technique are same as above.Determine whether the method for foreign aid's gene insertion: according to PHSE401 carrier sequence design primer, primer sequence are as follows:

II-F:5'-TTGTCACTGAAGCGGGAAG-3' of NPT；

II-R:5'-CCGTAAAGCACGAGGAAGC-3' of NPT；

PCR amplification is carried out to transgenic plant, judges T through 1.5% agarose gel electrophoresis₁Whether contain for mutant outer Help gene insertion.

It is analyzed according to sequencing result, finds M1 strain there are the missing of 1bp, there are the small fragment of 2bp missing, M3 for M2 strain There are the small fragments of 7bp to lack (Fig. 2) for strain.M1, M2 and M3 strain and wild-type tomatoes LA1996 strain are planted in modeling Expect in greenhouse (Guangzhou), in fruit green ripe stage and red ripe phase, all transgenic positive plant tomato leaf anthocyanidin contents with Wild-type tomatoes are not significantly different；All transgenic positive plant strains tamato fruit presentation atropurpureus, and wild type kind A little purple dot is only presented in fact in solanberry, and Study on Content Analysis of Anthocyanidin shows that anthocyanidin contains in all transgenic positive fruits/plants Measure it is extremely significant be higher than wild-type tomatoes LA1996, the results are shown in Table 1 and Fig. 3.Wherein, anthocyanidin content method for measuring is with reference to text Offer " Transcriptional analysis in high-anthocyanin tomatoes reveals synergistic effect of Aft and atv genes.《Journal of Plant Physiology》,2011,168(3):270- 279.”。

1 wild-type tomatoes of table and SlMYBATV mutant tomato anthocyanidin content (mg/100g FW)

Position	WT	M1	M2	M3
					Blade	2.85±0.34	2.45±0.32	2.93±0.29	3.04±0.35
Exocarp (green ripe stage)	13.00±1.98	314.42±24.38	348.259±29.98	286.69±20.25
					Exocarp (red ripe phase)	11.00±1.76	244.42±38.32	308.259±28.74	266.69±25.32

The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention, It should be equivalent substitute mode, be included within the scope of the present invention.

Sequence table

<110>Agricultural University Of South China

<120>a kind of methods and applications with CRISPR/Cas9 gene editing system initiative purple fruit Tomato mutants

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<211> 4081

<212> DNA

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<220>

<223>tomato SlMYBATV gene order

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atggcagatt ggaatagatc aagcacatca gataatgcct cagtggtctc acctggtaat 60

taacaattct tttttatttt atcgcaagag cttttaatta ttcttttcgt tcacttttat 120

tgatctattt tggacatctc atgctcctta ccaaaaattt cttttgatat aagggtccta 180

cagtacatat taattgatat ttgatattat gttttgaaag aagatttgag aaataaataa 240

ctaatgagct aagggtaaac atgaaataaa atatttgtct ttttttcatg ttaaaaagta 300

acgagtaaaa atgaacggat acttttatat ttgcttataa atatattcct taaaggaatt 360

tggagaacat tatgatatga ttatctgcgt ctaaccatat actctaataa tgccataagt 420

aaacaaatat ttatcctttt ggctacttcc aaaatacatg ttcatttatg aaatcatttt 480

ttttaataat aagttagtta gtcggaattt agaatttaaa atttatgtat ttttatacat 540

caagttaata tattacacta cttataagtt cacaattaaa tattcaattt tgttaataat 600

tttcttaata tatttataag tctaaataaa agttattgag ttcacgtgaa ttcattatag 660

attcgacccg agtggttgca ttagagacta ccaacgaaga aacctctaaa cttgaatttt 720

cagaagatga agaaatgctc attgctaaaa tgttcagctt ggttagagag aggtacgttt 780

aatatttttt taaaaaaatt cttgaatttt gtgtattaat tatttaaggt ttaatataaa 840

attgtcatgg tgtaatttta attaggtggt cattaattgc tggaagaatc ccaggaagaa 900

atgctgatga gattgaaaaa tattggaaat caaaatactc caaaagccag taaatttctt 960

caaattactt tttccctcag atgaatattt ttatctattt ttacttatct aatattaatt 1020

cgatgtactt cataatattt ttaaaaattt catagtaaat aacaaagata aataataagt 1080

gataagctta tctctagatt tctaagttgg ataagtaaga atgttgtaga aactagatag 1140

gaagtagttc tttcgttcac ttctatttat ctaacttatc tactgtagat tataattttt 1200

tttaaaaata cttttaaaac ttatttttca aattattttt caagacttag agattaaaat 1260

caatatatat atttaacata gggtagagga caaaaatcac atacttttaa ggtaaaatta 1320

ctatttgttc cttgtaaatt tataattaca aatatctctc aaactgatac aataatacaa 1380

gcgcgaatac attattcgtt aggtaagata caataaattt tatatacact gatctaatgt 1440

atattttata catgatacac taatctgatg cgcaagatac attaatatga agcgtgaaaa 1500

tgttaaattc gtaatcgtaa acaaacttga aactccgtcc cttgtggttt atgtactttc 1560

agaagttcgt gtatcttgca tgaaatttaa tatttttatt atttgaacag tatacacagt 1620

gaatatcatg tcatttcagg atcttcacat ttactttatt tttactttat ctacttttaa 1680

tttaacgatc cttagaaaaa atgatataat tattttatga taatatttgt attaattgat 1740

gtttaatatt atcttttgaa aaataattta gaaaataagt aattaatgtt aagaaaaaaa 1800

tcaaatattt ttatacgtta aaagtgacaa gtaaatgcaa ataaaaagta ttattcacta 1860

gatatcaata agcgagtaaa aatattcagt aagaagattc aacatatatt atatatatga 1920

acataaaaaa aaatttcaat cctttttgat tctttggtga cttaaactta catccttagt 1980

atattgattg tttaccattc gagcaacttc catgaaaatc ttttcattat ctgtaacgtt 2040

tttattagga gtttttttaa aaaaaataat aataaaaaca aaaatttgta ctcttcattt 2100

agggaggaaa tacaggccca atctcaagat gaagctcatg gcattagatt gatagagaaa 2160

actgggcctt caacggggca ccctcagcag tgtgtttttc cagaaaaaaa gcccatatct 2220

gtctgtcaac cttcagatat tgggcctcaa ctattgggcc caacaacgaa tgggcctcaa 2280

cagctattgg gccctaaaac aactgggccc aataacatca ctgtagacca aaagttagtt 2340

aacccagccc aagaccaaat aaaccaataa cctatgccca gcccagccca agattggtca 2400

aacatgacac tagacaaacc tacaatatat tttctattta catattttcg caaataccta 2460

ctactcaatt atgctatgta tagctatagt ttgcatattt actagttgta gttacagttt 2520

aagttatctc gtttgtataa tttccatata tgtttaaata agtgagttat atttgtataa 2580

actcaaaata atgaatttat acaaacacaa acatttaaac tttaaatagt tgtacagata 2640

tattatacaa aagttataca aattatatta tacaactttc caattataca cactcgattt 2700

gattttcttg gaagtgtttg acgacacaat tgtttatata gaccatgatc atgatcataa 2760

tatttgtgta tttgattaaa cttataatgt tattaaatta atttatacgt actttttaat 2820

aagttacaga tatctcatat cctttctaat aaaatatctt atatcctttc taataacctc 2880

tcctcgaaca attgctttaa gtttcaaaaa ttgactattt cctatttcat tttttgataa 2940

ttcctattaa gggaaaatta cgtggaatga caaacattcc taagtaatta gtcaataagg 3000

gtatatttta atttattgac tcaataacta tagtttcatt tttttgccat aattaatggg 3060

acccaccatc tctttgtata tccaaccatg aaacaaaaag ttatcttttt atatataatt 3120

ttatcaaatc aattttatct ctcctacact cattctatat ctttcttcta aaatcactcc 3180

tccactccta ttttgaattt tgaattttgg aaaatttcac catatccctg ttgtcttact 3240

ttcattaatc caattttttt ttaaaaaatt tcttcatcaa cacaattata tatgtattat 3300

ttttcttagt ttttttcata tattttttta aaaaaattct ttctttcttt tcagtttaaa 3360

gttcttcaaa atgagaataa agaattcatc tccatatatg agtattagca taggtatgtc 3420

attacatgtc aatattgttg ataatccacc ttcgttttca attgagttat ttcaggattt 3480

tggggtcaat gtagggtata tgacaaaacc taaaaaagtt caagttgaac aatatcttga 3540

atagagattc aagaaaaaaa tgacccaatt acaattcaga aagtcatcaa caacgcgaaa 3600

tctagcggca ttatagttga aggttgaagc aagagaaaag tcaaacataa attcagacga 3660

gatcggatct atctcatcaa aactggacag aactagacaa atctgaagaa catcaggttg 3720

tgtaatttat atacaaatta ctaaagttat gtatatagtg ggttgtgtat taaggtaata 3780

tatacaaagt attatgagat aattgttatt tgtatacaaa tatttgtata aaagaaattt 3840

gttaatcatt gtataataca tatataaaat tatagtacat tatacaactt tgtcgatata 3900

taaaaaaaat tatgaacatg ataaaatatt gtatatataa acattacaca ctaaatacac 3960

aaacatattt caagtttacc atatatacaa ttgacaaacg aatattatac aatcattttg 4020

atcattaatt tttttttaaa aacatgaaat tacatatgta catatacaaa tatgattcat 4080

a 4081

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<211> 19

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

The sequence of<223>first gRNA target sites

<400> 2

gagtggttgc attagagac 19

<210> 3

<211> 19

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

The sequence of<223>second gRNA target sites

<400> 3

acgaagaaac ctctaaact 19

<210> 4

<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

1. a kind of method with CRISPR/Cas9 gene editing technology initiative purple fruit Tomato mutants, it is characterised in that:

Including the design of gRNA target position on second exon of SlMYBATV gene, and two target site sequence spacing are no more than 100bp；

The sequence of first gRNA target site are as follows: 5'-gagtggttgcattagagac-3'；

The sequence of second gRNA target site are as follows: 5'-acgaagaaacctctaaact-3'；

Including gRNA PCR primer:

Upstream primer gRNA-F:5'-ATATATGGTCTCGTTTGgtctctaatgcaaccactcGTTTTAGAGC TAGAAAT AG-3'；

Downstream primer gRNA-R:5'-ATTATTGGTCTCGAAACacgaagaaacctctaaactCCAAACTACA CTGTTAGA TTC-3'。

2. according to claim 1 a kind of with the initiative purple fruit tomato mutation of CRISPR/Cas9 gene editing technology The method of body, which comprises the following steps:

(1) selection of gRNA target site: two target sites are designed on second exon of SlMYBATV gene, and two A target site sequence spacing is no more than 100bp；

(2) design of gRNA PCR primer:

Downstream primer gRNA-R:5'-ATTATTGGTCTCGAAACacgaagaaacctctaaactCCAAACTACA CTGTTAGA TTC-3'；

(3) building of gRNA expression vector:

Using plasmid pCBC-DT1T2 as template, PCR amplification, purifying are carried out with upstream primer gRNA-F and downstream primer gRNA-R, It obtains gRNA segment and purifies；Digestion CRISPR/Cas9 vector plasmid and the gRNA after purification are distinguished with restriction endonuclease Bsa I Segment；CRISPR/Cas9 vector plasmid and gRNA segment after connecting the digestion with T4 ligase after purification obtain connection and produce Object；By connection product conversion, screening, verify to get CRISPR/Cas9-gRNA expression vector；

(4) CRISPR/Cas9-gRNA expression vector is imported in Agrobacterium, obtains CRISPR/Cas9-gRNA Agrobacterium；With CRISPR/Cas9-gRNA Agrobacterium infects the wild-type tomatoes LA1996 callus containing the site Aft；

(5) callus for obtaining step (4), receives chloramphenicol resistance screening through card, and induction obtains obtaining through screening after regrowth Transgenic positive plant；

(6) it after obtaining positive transgenic strain, is verified through the sequencing of gene target site, foreign aid's gene insertion analysis and mutation type surface For positive homozygous lines, SlMYBATV function and the purple fruit tomato mutation without the insertion of foreign aid's gene are as completely lost Body.

3. according to claim 2 a kind of with the initiative purple fruit tomato mutation of CRISPR/Cas9 gene editing technology The method of body, it is characterised in that:

CRISPR/Cas9 carrier described in step (3) is pHSE401.

4. according to claim 2 a kind of with the initiative purple fruit tomato mutation of CRISPR/Cas9 gene editing technology The method of body, it is characterised in that:

PCR amplification system described in step (3) are as follows: 1 μ L, KAPA HiFi HotStart ReadyMix of pHSE401 plasmid 25 22 μ L of μ L, gRNA-R of μ L, gRNA-F, add ddH₂O to 50 μ L；PCR amplification program are as follows: 95 DEG C of initial denaturation 3min；95 DEG C of changes Property 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 1min, 32 circulation；72 DEG C of extension 5min.

5. according to claim 2 a kind of with the initiative purple fruit tomato mutation of CRISPR/Cas9 gene editing technology The method of body, it is characterised in that:

GRNA segment digestion system described in step (3) are as follows: 2 μ g, 10 × CutSmart Buffer of gRNA segment 5 μ L, Bsa I 10U, adds ddH₂O to 50 μ L；CRISPR/Cas9 vector plasmid digestion system are as follows: CRISPR/Cas9 vector plasmid 2 μ g, 10 × 5 I 10U of μ L, Bsa of CutSmart Buffer, adds ddH₂O to 50 μ L.

6. according to claim 2 a kind of with the initiative purple fruit tomato mutation of CRISPR/Cas9 gene editing technology The method of body, it is characterised in that:

Digestion condition described in step (3) are as follows: 37 DEG C of digestion 2h.

7. according to claim 2 a kind of with the initiative purple fruit tomato mutation of CRISPR/Cas9 gene editing technology The method of body, it is characterised in that:

T4 ligase linked system described in step (3) are as follows: digestion, 8 μ L of CRISPR/Cas9 vector plasmid after purification, enzyme It cuts, 4 μ L, 10 × T4 DNA ligase buffer of gRNA segment, 5 μ L, T4 DNA ligase, 1 μ L after purification, adds ddH₂O to 50 μ L。

8. according to claim 2 a kind of with the initiative purple fruit tomato mutation of CRISPR/Cas9 gene editing technology The method of body, it is characterised in that:

9. according to claim 2 a kind of with the initiative purple fruit tomato mutation of CRISPR/Cas9 gene editing technology The method of body, it is characterised in that:

Agrobacterium described in step (4) is Agrobacterium tumefaciems AGL1.

10. according to any one of claims 1 to 9 a kind of with CRISPR/Cas9 gene editing technology initiative purple fruit kind Application of the purple fruit Tomato mutants of the method preparation of eggplant mutant in tomato breeding.