阅读说明：本技术 一种应用于马铃薯上的CRISPR/Cas9载体的构建方法 (Construction method of CRISPR/Cas9 vector applied to potatoes ) 是由 闫建俊 白云凤 左静静 左敏 李萌 于 2019-09-25 设计创作，主要内容包括：本发明公开了一种应用于马铃薯上的CRISPR/Cas9载体的构建方法,属于植物分子生物学领域。该方法包括如下步骤：根据马铃薯中糖苷生物碱合成关键酶基因PGA2设计用于基因敲除的gRNA靶点,合成引物gRNA,然后将gRNA和Cas9蛋白结合形成表达盒,再将表达盒插入到表达载体PCAMBIA 1300中,将得到的连接物转化到大肠杆菌中,摇菌测序后,构建用于遗传转化的农杆菌工程菌,转化马铃薯,提取基因组DNA,筛选出阳性植株。本发明首次在马铃薯作物中通过CRISPR/Cas9基因编辑技术对马铃薯中糖苷生物碱合成关键酶基因进行编辑,为后续基因功能研究或马铃薯基因工程育种等奠定技术基础。(The invention discloses a construction method of a CRISPR/Cas9 vector applied to potatoes, belonging to the field of plant molecular biology. The method comprises the following steps: designing a gRNA target point for gene knockout according to a glucoside alkaloid synthesis key enzyme gene PGA2 in the potato, synthesizing a primer gRNA, combining the gRNA and Cas9 protein to form an expression cassette, inserting the expression cassette into an expression vector PCAMBIA1300, transforming the obtained connector into escherichia coli, shaking the bacteria for sequencing, constructing agrobacterium engineering bacteria for genetic transformation, transforming the potato, extracting genome DNA, and screening out positive plants. The invention edits the glucoside alkaloid synthesis key enzyme gene in the potato by the CRISPR/Cas9 gene editing technology for the first time in potato crops, and lays a technical foundation for subsequent gene function research or potato gene engineering breeding and the like.)

1. A construction method of a CRISPR/Cas9 vector applied to potatoes is characterized by comprising the following steps:

s1, CRISPR target design:

sgRNA was designed according to sequence number AB839753.1 of potato PGA2 gene: GGTGTGAAATCGTGGTGCAATGG, respectively;

s2, designing a primer oligo by utilizing the principle of homologous recombination:

forward sequence: 5'-AGTCGAAGTAGTGATTGGGGTGTGAAATCGTGGTGCAA-3' the flow of the air in the air conditioner,

reverse sequence: 5'-ATTTCTAGCTCTAAAACCTTGCACCACGATTTCACACC-3', respectively;

s3, formation of primer gRNA:

respectively diluting the synthesized primer oligos to 10 mu M, mixing the oligos according to the following proportion, and carrying out annealing reaction to form a primer gRNA;

reaction conditions are as follows: at 95 ℃ for 2 min; at 95 ℃ for 20 s; 60 ℃ for 40 s; 72 ℃ for 15 s; 15 cycles; 72 ℃ for 5 min;

s4, combining gRNA and Cas9 protein to form an expression cassette, connecting the expression cassette into a vector psgR-Cas9-At to form an intermediate vector, performing double enzyme digestion on the intermediate vector by using EcoRI and Hind III to obtain an enzyme digestion product as an Oligo primer dimer, performing double enzyme digestion on a PCAMBIA1300 vector by using EcoRI and Hind III to obtain an enzyme digestion linearized vector, mixing the enzyme digestion linearized vector according to the following proportion, and connecting the enzyme digestion linearized vector At 37 ℃ for 30min to obtain an editing vector;

s5, transforming the editing vector obtained in the step S4 into escherichia coli;

s6, picking 5 single colonies, shaking bacteria and sequencing;

s7, transferring the successfully constructed vector into agrobacterium to construct engineering bacteria, and transferring the engineering bacteria into potatoes through genetic transformation.

2. The method for constructing the CRISPR/Cas9 vector for potato application according to claim 1, wherein in step S1, the sgRNA sequence is GGTGTGAAATCGTGGTGCAATGG, GGTGTGAAATCGTGGTGCAA which is a gRNA target designed for gene knockout, and the TGG is a PAM sequence.

3. The method for constructing the CRISPR/Cas9 vector applied to potatoes as claimed in claim 1, wherein the S5 comprises the specific steps of adding 10 μ L of the editing vector into 50 μ L of Top10 competent cells, mixing, performing heat shock transformation, smearing on a culture medium plate containing kanamycin, and culturing overnight at 37 ℃.

4. The method for constructing the CRISPR/Cas9 vector applied to potatoes as claimed in claim 1, wherein the specific steps of S7 are as follows:

s71 expanding propagation of aseptic seedlings

The stem section propagation culture medium is an MS solid culture medium; cutting stem sections of the aseptic seedlings into about 2cm and inserting the stem sections into a culture medium during propagation of the aseptic seedlings;

s72, agrobacterium propagation

Streaking glycerol bacteria on an LB solid culture medium, culturing for two days at 28 ℃, selecting a monoclonal antibody, inoculating the monoclonal antibody into 5ml of an LB liquid culture medium, carrying out constant-temperature shaking culture at 28 ℃, 220rpm and overnight till OD600 is about 0.6, centrifuging at 5000rpm for 10min, collecting the bacteria, and re-suspending the collected bacteria by using an MS liquid culture medium until OD600 is 0.4;

s73, agrobacterium infection and co-culture

Cutting the stem segment of the aseptic seedling into about 2cm, placing the stem segment into the resuspended agrobacterium liquid, gently shaking for 10min, sucking the liquid on the surface of the stem segment by using aseptic filter paper, and transferring the stem segment onto a co-culture medium for dark culture for 2-3 days;

s74 bud-induced differentiation

After co-culture, transferring the explant to a bud induction differentiation culture medium, inducing germination, and replacing the culture medium every two weeks;

s75 rooting and resistant plant screening

When the resistant bud grows to 2cm, cutting the bud and transferring the bud into a rooting culture medium, cutting partial leaves when a complete plantlet is formed, extracting genome DNA by using a full-type gold plant tissue direct amplification kit, and designing a specific primer to detect the plantlet;

s76 PCR amplification

Mixing the extracted genome DNA according to the following proportion, uniformly mixing, and carrying out electrophoresis on the size of a PCR product by using 1.0% agarose gel to obtain the PCR product;

reaction conditions are as follows: 5min at 94 ℃; 30s at 94 ℃; 30s at 58 ℃; 30s at 72 ℃; 30 cycles; 5min at 72 ℃.

5. The construction method of CRISPR/Cas9 vector applied on potato according to claim 1,

the MS culture medium comprises: 4.42g/LMS, 3% sucrose, 7g/L agar powder, PH 5.8;

the LB culture medium comprises: 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride, 50mg/L kanamycin and 50mg/L gentamicin;

the co-culture medium comprises: MS, 1.0mg/L indoleacetic acid, 0.2mg/L gibberellin, 2.0mg/L zeatin and 0.5mg/L cytokinin, wherein the PH is 5.8;

the bud induction differentiation culture medium comprises: MS, 1.0mg/L indoleacetic acid, 0.2mg/L gibberellin, 2.0mg/L zeatin, 0.5mg/L cytokinin, 50mg/L kanamycin and 500mg/L thielavonoid, wherein the PH is 5.8;

the rooting culture medium comprises: MS, 50mg/L kanamycin, pH 5.8.

Technical Field

The invention relates to the field of plant molecular biology, in particular to a construction method of a CRISPR/Cas9 vector applied to potatoes.

Background

Genome editing is a genetic manipulation technique that can modify DNA sequences at the genome level, also known as gene targeting. The technical principle is that an artificial nuclease is constructed, DNA is cut off at a preset genome position, and the cut DNA generates mutation in the process of being repaired by a DNA repair system in a cell, so that the aim of site-specific genome modification is fulfilled. And the CRISPR-Cas9 (CRISPR) gene editing technology is the most popular gene editing technology at present due to the simplicity and low cost. Since the advent of this technology, there have been incomparable advantages over other gene editing technologies, and it is believed that the technology will be able to "edit" any gene most efficiently and conveniently in living cells, with continued improvement. Under the guide RNA (sgRNA), a target sequence near PAM (protospacer adjacent motif) can be targeted through base complementary pairing, and the Cas9 protein is a double-stranded DNA nuclease and can cut a target point on the upstream and downstream of the gene to break a DNA double strand. And the organism has a response mechanism of DNA damage repair, and sequences at two ends of the upstream and the downstream of the fracture can be connected, so that the target gene in the cell can be knocked out. The technology has wide application prospect in the aspects of bioengineering and the like, and is already applied to a plurality of crops.

The potato has anti-aging effect. It contains rich vitamin B1, vitamin B2, vitamin B6, pantothenic acid and other vitamins, great amount of high quality cellulose, trace elements, amino acids, protein, fat, high quality starch and other nutritious elements. In recent years, with the improvement of living standard of people, the requirements on the quality of potatoes are higher and higher, and the CRISPR/Cas9 gene editing technology is utilized to play an innovative role in improving the quality of potatoes.

Disclosure of Invention

The invention aims to use a CRISPR/Cas9 mediated plant gene editing vector in the research of potato gene function or the application of potato gene engineering breeding.

In order to achieve the purpose, the invention provides the following technical scheme:

a construction method of a CRISPR/Cas9 vector applied to potatoes comprises the following steps:

s1, CRISPR target design:

sgRNA was designed according to sequence number AB839753.1 of potato PGA2 gene: GGTGTGAAATCGTGGTGCAATGG, respectively;

s2, designing a primer oligo by utilizing the principle of homologous recombination:

forward sequence: 5'-AGTCGAAGTAGTGATTGGGGTGTGAAATCGTGGTGCAA-3' the flow of the air in the air conditioner,

reverse sequence: 5'-ATTTCTAGCTCTAAAACCTTGCACCACGATTTCACACC-3', respectively;

s3, formation of primer gRNA:

respectively diluting the synthesized primer oligos to 10 mu M, mixing the oligos according to the following proportion, and carrying out annealing reaction to form a primer gRNA;

reaction conditions are as follows: at 95 ℃ for 2 min; at 95 ℃ for 20 s; 60 ℃ for 40 s; 72 ℃ for 15 s; 15 cycles; 72 ℃ for 5 min;

s4, combining gRNA and Cas9 protein to form an expression cassette, connecting the expression cassette into a vector psgR-Cas9-At to form an intermediate vector, performing double enzyme digestion on the intermediate vector by using EcoRI and Hind III to obtain an enzyme digestion product as an Oligo primer dimer, performing double enzyme digestion on a PCAMBIA1300 vector by using EcoRI and Hind III to obtain an enzyme digestion linearized vector, mixing the enzyme digestion linearized vector according to the following proportion, and connecting the enzyme digestion linearized vector At 37 ℃ for 30min to obtain an editing vector;

s5, transforming the editing vector obtained in the step S4 into escherichia coli;

s6, picking 5 single colonies, shaking bacteria and sequencing;

s7, transferring the successfully constructed vector into agrobacterium to construct engineering bacteria, and transferring the engineering bacteria into potatoes through genetic transformation.

Preferably, in step S1, the sgRNA sequence GGTGTGAAATCGTGGTGCAATGG, GGTGTGAAATCGTGGTGCAA is a gRNA target designed for gene knockout, and the TGG is a PAM sequence.

Preferably, the specific step of S5 is to take 10. mu.L of the editing vector, add it to 50. mu.L of Top10 competent cells, mix them well, heat shock transform, smear it on a kanamycin-containing medium plate, and culture it overnight at 37 ℃.

Preferably, the specific steps of S7 are as follows:

s71 expanding propagation of aseptic seedlings

The stem section propagation culture medium is an MS solid culture medium; cutting stem sections of the aseptic seedlings into about 2cm and inserting the stem sections into a culture medium during propagation of the aseptic seedlings;

s72, agrobacterium propagation

Streaking glycerol bacteria on an LB solid culture medium, culturing for two days at 28 ℃, selecting a monoclonal antibody, inoculating the monoclonal antibody into 5ml of an LB liquid culture medium, carrying out constant-temperature shaking culture at 28 ℃, 220rpm and overnight till OD600 is about 0.6, centrifuging at 5000rpm for 10min, collecting the bacteria, and re-suspending the collected bacteria by using an MS liquid culture medium until OD600 is 0.4;

s73, agrobacterium infection and co-culture

Cutting the stem segment of the aseptic seedling into about 2cm, placing the stem segment into the resuspended agrobacterium liquid, gently shaking for 10min, sucking the liquid on the surface of the stem segment by using aseptic filter paper, and transferring the stem segment onto a co-culture medium for dark culture for 2-3 days;

s74 bud-induced differentiation

After co-culture, transferring the explant to a bud induction differentiation culture medium, inducing germination, and replacing the culture medium every two weeks;

s75 rooting and resistant plant screening

When the resistant bud grows to 2cm, cutting the bud and transferring the bud into a rooting culture medium, cutting partial leaves when a complete plantlet is formed, extracting genome DNA by using a full-type gold plant tissue direct amplification kit, and designing a specific primer to detect the plantlet;

s76 PCR amplification

Mixing the extracted genome DNA according to the following proportion, uniformly mixing, and carrying out electrophoresis on the size of a PCR product by using 1.0% agarose gel to obtain the PCR product;

reaction conditions are as follows: 5min at 94 ℃; 30s at 94 ℃; 30s at 58 ℃; 30s at 72 ℃; 30 cycles; 5min at 72 ℃.

In particular, the amount of the solvent to be used,

the MS culture medium comprises: 4.42g/LMS, 3% sucrose, 7g/L agar powder, PH 5.8;

the LB culture medium comprises: 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride, 50mg/L kanamycin and 50mg/L gentamicin;

the co-culture medium comprises: MS, 1.0mg/L indoleacetic acid, 0.2mg/L gibberellin, 2.0mg/L zeatin and 0.5mg/L cytokinin, wherein the PH is 5.8;

the bud induction differentiation culture medium comprises: MS, 1.0mg/L indoleacetic acid, 0.2mg/L gibberellin, 2.0mg/L zeatin, 0.5mg/L cytokinin, 50mg/L kanamycin and 500mg/L thielavonoid, wherein the PH is 5.8;

the rooting culture medium comprises: MS, 50mg/L kanamycin, pH 5.8.

Compared with the prior art, the invention has the advantages that: the gene editing vector is obtained in the potato through the CRISPR/Cas9 gene editing technology for the first time, and a technical basis is laid for subsequent gene function research or potato gene engineering breeding and the like.

Drawings

FIG. 1 is a map of a successfully constructed gene editing vector;

FIG. 2 shows the result of PCR detection of genome DNA of a part of transgenic potato regenerated plants.

Detailed Description

The preferred embodiments of the present invention will be described in conjunction with the accompanying drawings, and it will be understood that they are described herein for the purpose of illustration and explanation and not limitation.

A construction method of a CRISPR/Cas9 vector applied to potatoes comprises the following steps:

s1, CRISPR target design:

sgRNA was designed according to potato PGA gene sequence number AB 839753.1: GGTGTGAAATCGTGGTGCAATGG, respectively;

s2, designing a primer oligo by utilizing the principle of homologous recombination:

forward sequence: AGTCGAAGTAGTGATTGGGGTGTGAAATCGTGGTGCAA the flow of the air in the air conditioner,

reverse sequence: ATTTCTAGCTCTAAAACCTTGCACCACGATTTCACACC, respectively;

s3, formation of primer gRNA:

respectively diluting the synthesized primer oligos to 10 mu M, mixing the oligos according to the following proportion, and carrying out annealing reaction to form a primer gRNA;

forward primer	1μL
		Reverse primer	1μL
2*fast pfu mix	12.5μL
		ddH2O	10.5μL
Total volume	25μL

Reaction conditions are as follows: at 95 ℃ for 2 min; at 95 ℃ for 20 s; 60 ℃ for 40 s; 72 ℃ for 15 s; 15 cycles; 72 ℃ for 5 min;

s4, combining gRNA and Cas9 protein to form an expression cassette, connecting the expression cassette into a vector psgR-Cas9-At to form an intermediate vector, performing double enzyme digestion on the intermediate vector by using EcoRI and Hind III to obtain an enzyme digestion product as an Oligo primer dimer, performing double enzyme digestion on a PCAMBIA1300 vector by using EcoRI and Hind III to obtain an enzyme digestion linearized vector, mixing the enzyme digestion linearized vector according to the following proportion, and connecting the enzyme digestion linearized vector At 37 ℃ for 30min to obtain an editing vector;

oligo primer dimer	8μL
		Enzyme digestion linearized vector	5μL
Recombinant ligase	1μL
		Recombinant ligase Buf.	2μL
ddH2O	4μL
		Total volume	20μL

S5, 10. mu.L of the editing vector obtained in step S4 was added to 50. mu.L of Top10 competent cells, mixed well, heat-shocked to transform, smeared on an LB medium plate containing kanamycin, and cultured overnight at 37 ℃.

S6, picking 5 single colonies, shaking bacteria and sequencing the vector, wherein the sequencing result is as follows:

TTTTCGTTTGAAACGGAACTCGACTTGCCTTCCGCACAATACATCATTTCTTCTTAGCTTTT TTTCTTCTTCTTCGTTCATACAGTTTTTTTTTGTTTATCAGCTTACATTTTCTTGAACCGTA GCTTTCGTTTTCTTCTTTTTAACTTTCCATTCGGAGTTTTTGTATCTTGTTTCATAGTTTGT CCCAGGATTAGAATGATTAGGCATCGAACCTTCAAGAATTTGATTGAATAAAACATCTTCAT TCTTAAGATATGAAGATAATCTTCAAAAGGCCCCTGGGAATCTGAAAGAAGAGAAGCAGGCC CATTTATATGGGAAAGAACAATAGTATTTCTTATATAGGCCCATTTAAGTTGAAAACAATCT TCAAAAGTCCCACATCGCTTAGATAAGAAAACGAAGCTGAGTTTATATACAGCTAGAGTCGA AGTAGTGATTGGGGTGTGAAATCGTGGTGCAAGGTTTTAGAGCTAGAAATAGCAAGTTAAAA TAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTGTTTTAGAG CTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTTTTAGCGCGTGCATGCCTGCAGGTGAG ACTTTTCAACAAAGGGTAATATCGGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCA CTTCATCAAAAGGACAGTAGAAAAGGAAGGTGGCACCTACAAATGCCATCATTGCGATAAAG GAAAGGCTATCGTTCAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACG AGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGA TATCTCCACTGACGTACGGGATGACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCTA TATAAGGAAGTC；

s7, transferring the successfully constructed vector engineering bacteria into potatoes, and specifically comprising the following steps:

s71 expanding propagation of aseptic seedlings

The stem segment propagation culture medium is MS solid culture medium (containing 3% sucrose, pH 5.8); when the aseptic seedling is propagated, the stem section of the aseptic seedling is cut into about 2cm and inserted into a culture medium.

S72, agrobacterium propagation

Glycerol bacteria were streaked on LB solid medium (50mg/L kanamycin +50mg/L gentamicin), after two days of culture at 28 ℃, single colonies were picked up and inoculated into 5ml of LB liquid medium (containing 50mg/L kanamycin +50mg/L gentamicin), incubated overnight at 28 ℃ and 220rpm with constant temperature shaking until OD600 became about 0.6, and then centrifuged at 5000rpm for 10min to collect the bacteria, and the collected bacteria were resuspended in MS liquid medium until OD600 became 0.4.

S73, agrobacterium infection and co-culture

The stem segment of the aseptic seedling is cut into about 2cm, placed in the resuspended agrobacterium liquid, gently shaken for 10min, the bacterial liquid on the surface of the stem segment is sucked up by aseptic filter paper, and the stem segment is transferred to a co-culture medium (MS +1.0mg/L indoleacetic acid +0.2mg/L gibberellin +2.0mg/L zeatin +0.5mg/L cytokinin, PH ═ 5.8) to be cultured in the dark for 2-3 days.

S74 bud-induced differentiation

After co-culture, explants were transferred to shoot-inducing differentiation medium (MS +1.0mg/L indoleacetic acid +0.2mg/L gibberellin +2.0mg/L zeatin +0.5mg/L cytokinin +50mg/L kanamycin +500mg/L thienamycin, pH 5.8) to induce shoots, and the medium was changed every two weeks.

S75 rooting and resistant plant screening

When the resistant bud grows to 2cm, cutting the bud and transferring the bud into a rooting culture medium (MS +50mg/L kanamycin, pH 5.8), when a complete plantlet is formed, cutting partial leaves, extracting genome DNA by using a full-scale gold plant tissue PCR Kit, and designing a specific primer to detect the plantlet.

S76 PCR amplification

Mixing the extracted genome DNA according to the following proportion, and reacting under the condition: 5min at 94 ℃; 30s at 94 ℃; 30s at 58 ℃; 30s at 72 ℃; 30 cycles; the PCR product is obtained by electrophoresis of 1.0 percent agarose gel at 72 ℃ for 5 min;

detecting the primer sequence, and screening out positive plants, as shown in FIG. 2, wherein the polymerase chain reaction detection result shows that lanes 1-16 are transgenic regeneration plants, negative is non-transgenic negative control plants, positive is transgenic positive control plants, and double distilled water is blank control; the PCR product size in the positive plant is 499bp, and the detected primer sequence is as follows:

forward sequence: 5'-CAGGAAACAGCTATGAC-3'

Reverse sequence: 5'-TTGCACCACGATTTCACACC-3'

Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.