CN114606254B - Construction method of vibrio parahaemolyticus gene efficient knockout plasmid - Google Patents

Abstract

The application discloses a construction method of a vibrio parahaemolyticus gene efficient knockout plasmid, belonging to the fields of molecular biology and biotechnology. The application optimizes the knocking-out process and introduces a Cre/loxP system. A gentamicin resistance fragment with loxP site was added between knockout homology arms, and then the resistance fragment was removed by Cre enzyme. Gentamicin is added during the second homologous recombination, and only strains which are subjected to the homologous recombination correctly can survive on a 10% sucrose gentamicin resistance plate, so that the screening efficiency of knockout strains is improved. Then, the second binding transduction was performed, and the pOTC plasmid with cre gene was introduced into Vibrio parahaemolyticus. The Cre gene expresses Cre enzyme in cells, recognizes loxP site, and removes gene segment between loxL and loxR, namely removes gentamicin resistance segment, and removes pOTC plasmid in test tube containing 10% sucrose.

Description

Construction method of vibrio parahaemolyticus gene efficient knockout plasmid

Technical Field

The application relates to a construction method of a vibrio parahaemolyticus gene efficient knockout plasmid, belonging to the fields of molecular biology and biotechnology.

Background

The traditional knockout flow of the vibrio parahaemolyticus is as follows: the homology arm was linked to a suicide plasmid, transduced into cells by conjugation and integrated into the genome by homologous recombination. Next, in a 10% sucrose environment, sacB integrated onto the plasmid in the genome will break down the sucrose to produce a lethal substance, thereby forcing the cells to undergo a second homologous recombination to remove the plasmid. Two conditions can occur in the second homologous recombination, wherein the first is that the homologous recombination occurs at a position different from that of the first step, namely, the homologous recombination of the first step and the second step occurs on an upstream/downstream homology arm respectively, so that the target gene fragment is removed along with the removal of the plasmid, and the knockout is successful; the second case is that homologous recombination occurs at the same position as in the first step, i.e., homologous recombination occurs in the same homology arm as in the second step, with the result that the plasmid integrated in the genome is removed intact and the knockout fails. With the importance of the target gene in the cell, the ease of knockout will vary. In some gene knockout processes, the second step can take a significant amount of time and resources.

Further, since mutation of sacB gene is liable to occur and false positive rate of reverse screening is high, it is necessary to identify a large number of mutants in the end, and a large amount of time and resources are required. Therefore, there is an urgent need for a method of knocking out Vibrio parahaemolyticus with high accuracy.

Disclosure of Invention

In order to solve the technical problems, the application optimizes the knockout process and introduces a Cre/loxP system. A gentamicin resistance fragment with loxP site was added between knockout homology arms, and then the resistance fragment was removed by Cre enzyme. Gentamicin is added during the second homologous recombination, and only strains which are subjected to the homologous recombination correctly can survive on a 10% sucrose gentamicin resistance plate, so that the screening efficiency of knockout strains is improved. And then carrying out the second binding transduction, and introducing the pOTC plasmid with the Cre gene into the vibrio parahaemolyticus. The Cre gene expresses Cre enzyme in cells, recognizes loxP site, and removes gene fragment between loxL and loxR, i.e., removes gentamicin resistance fragment. Finally, the pOTC plasmid was removed in tubes containing 10% sucrose.

The application provides a knockout plasmid pOTC, which comprises a replicon p15A, a resistance gene CmR, transfer elements oriT and traJ, a promoter Ptac and a Cre gene; the nucleotide sequence of the knocked-out plasmid pOTC is shown as SEQ ID NO. 1.

In one embodiment of the present application, the knockout plasmid pOTC further comprises a sacB gene; the nucleotide sequence of the SacB gene is shown as SEQ ID NO. 2.

The application also provides a method for knocking out a target gene in vibrio parahaemolyticus, which comprises the following steps:

(1) Synthesizing a loxL-Gm-loxR fragment;

(2) Integrating the homologous arm homoloxL-Gm-loxR fragment of the upstream and downstream of the target gene onto a pDS132 plasmid to obtain a recombinant plasmid pDS132-1;

(3) Introducing the recombinant plasmid pDS132-1 into the competence of the escherichia coli to obtain recombinant escherichia coli/pDS 132-1;

(4) Recombinant escherichia coli/pDS 132-1 and vibrio parahaemolyticus are combined and transduced to obtain recombinant vibrio parahaemolyticus/pDS 132-1 containing recombinant plasmid pDS132-1, and the recombinant vibrio parahaemolyticus/pDS 132-1 is inoculated into a sucrose culture medium containing gentamicin for culture;

(5) Introducing the knockout plasmid pOTC containing the SacB gene into escherichia coli competence to obtain recombinant escherichia coli/pOTC-SB; and (3) combining and transduction of recombinant escherichia coli/pOTC-SB and the recombinant vibrio parahaemolyticus cultured in the step (4), and inoculating the recombinant escherichia coli/pOTC-SB into a culture medium containing chloramphenicol for culture to obtain the vibrio parahaemolyticus from which the target gene is knocked out.

In one embodiment of the present application, in step (1), the nucleotide sequence of the loxL-Gm-loxR fragment is shown in SEQ ID NO. 3.

In one embodiment of the present application, in steps (3) and (5), the E.coli includes, but is not limited to, CC118 (λpir), S17-1 (λpir).

In one embodiment of the present application, in step (4), the sucrose medium containing gentamicin is an LB medium, and the sucrose is added in an amount of at least: 10% (w/v).

In one embodiment of the present application, the gentamicin is added in an amount of: 10-30 mg/L.

In one embodiment of the present application, the medium containing chloramphenicol is an LB medium, and the amount of chloramphenicol added is: 6-30 mg/L.

In one embodiment of the present application, the gene of interest is any gene on the genome of Vibrio parahaemolyticus that does not affect its growth.

In one embodiment of the application, the genes of interest include, but are not limited to, a protein with a gene accession number VP_RS18950 on NCBI, a protein with a gene accession number VP_RS17740 on NCBI, a protein with a gene accession number VP_RS16510 on NCBI, a protein with a gene accession number VP_RS06135 on NCBI, a protein with a gene accession number VP_RS02520 on NCBI, a protein with a gene accession number VP_RS11975 on NCBI, a protein with a gene accession number VP_RS22195 on NCBI, a protein with a gene accession number VP_RS23020 on NCBI, a protein with a gene accession number VP_RS16800 on NCBI, a protein with a gene accession number VP_RS16465 on NCBI, a gene bank RS 40 on NCBI, a gene accession number VP_RS 3898 on NCBI, and a gene accession number VP_RS 20828 on NCBI.

In one embodiment of the application, the rpoE gene has a gene bank accession number on NCBI of: vp_rs12550. Sigma expression of rpoE ^E Factors play an important role in bacterial extracellular pressure response (Hews C L, cho T, rowley G, et al, maintaining Integrity Under Stress: envelope Stress Response Regulation of Pathogenesis in Gram-Negative Bacteria [ J)]Front Cell Infect Microbiol,2019, 9:313.). rpoE is an essential gene in E.coli at high temperature (De Las Penas A, connolly L, gross C A. SigmaE is an essential sigma factor in Escherichia coli [ J)]J Bacteriol,1997,179 (21): 6862-6864.) the difficulty of knockout is greater.

In one embodiment of the present application, the Vibrio parahaemolyticus includes, but is not limited to, vibrio parahaemolyticus ATCC33846.

The application also provides application of the knockout plasmid in knocking out any gene which does not influence the growth of vibrio parahaemolyticus genome.

Advantageous effects

(1) The application provides a method for constructing vibrio parahaemolyticus knockout tool plasmid by using gene fragments on 3 existing vectors through overlapping PCR and one-step cloning, and the plasmid is used for constructing vibrio parahaemolyticus gene knockout strain and researching gene functions.

(2) The conventional vibrio parahaemolyticus knockout method has a knockout success probability of theoretically 50% at the time of the second homologous recombination. The application introduces the gentamicin resistance gene, adds gentamicin during the second homologous recombination, screens out the strain reverting to the wild type during the second homologous recombination through the resistance gene, improves the screening efficiency of correct strains, and screens out the reverting mutation with the efficiency of 100%. Meanwhile, the method can reduce the number and time of picking colonies, save the time of configuring a verification system, save the use amount of verification enzymes, and can quickly obtain the gene knockout mutant strain.

Drawings

Fig. 1: flow chart after optimizing the introduction of vibrio parahaemolyticus into the pOTC plasmid.

Fig. 2: construction flow chart of knockout plasmids pOTC and pOTC-SB.

Fig. 3: construction verification of pOTC and pOTC-SB; lanes 1 and 2 are BstZ17I, ecoRI digestion verification of pOTC respectively; bstZ17I, ecoRI cleavage of pOTC-SB in lanes 3 and 4, respectively; m represents a DNA marker of 250-10000 bp.

Fig. 4: agarose gel verification of the electropherogram after a second homologous recombination with the rpoE gene knocked out using an optimization system, wherein M represents a DNA Marker of 250-5000bp and C represents a colony.

Fig. 5: verifying the electrophoresis pattern by using agarose gel after the second homologous recombination of the rpoE gene knocked out by the original system; m represents a 250-10000bp DNA Marker, G represents a wild-type genome control, P represents an upstream and downstream homology arm control, and C represents a colony.

Fig. 6: panel streaking verifies removal of Gm gene after rpoE knockout.

Fig. 7: the removal of plasmid pOTC-SB after rpoE knockout was verified by plate streaking.

Fig. 8: the optimized vibrio parahaemolyticus knockout system is used for knocking out the outer membrane protein, and the graph shows the knocking-out result of agarose gel electrophoresis verification; wherein, lane 1 is 8 outer membrane protein genes in wild type ATCC33846, lanes 2-9 are in order Δvp_rs11975, Δvp_rs22195, Δvp_rs23020, Δvp_rs16800, Δvp_rs16465, Δvp_rs20840, Δvp_rs03765, Δvp_rs11205; m represents a DNA marker of 250-5000 bp.

Detailed Description

The application is further illustrated in the following drawings and specific examples, which are not intended to limit the application in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present application are those conventional in the art.

Unless otherwise indicated, the reagents and materials used in the following examples are commercially available or may be prepared by known methods.

The method of the present application is applicable to all of Vibrio parahaemolyticus, and the following examples are given by way of example only of Vibrio parahaemolyticus ATCC33846, but are not intended to limit the scope of the present application.

The following examples relate to the following media:

ddH was used for the medium ₂ And (3) preparing O, and sterilizing for 15-20 min at 121 ℃ after the preparation is finished.

LB medium (g/L): yeast powder 5, peptone 10 and NaCl 10.

The primer sequences involved in the following examples are shown in Table 1:

TABLE 1 primer sequences according to the following examples

The sources of the strains and plasmids referred to in the examples below are shown in Table 2:

TABLE 2 strains and plasmids involved in the examples below

Example 1: construction of knockout plasmid

The method comprises the following specific steps:

1. construction of plasmid pOTC

(1) The fragment containing the CmR and p15A genes was amplified from pACYC184 using the primers Cm-p15A+ and Cm-p15A-. The PCR product was recovered using a PCR product recovery kit and digested with SpeI.

(2) The traJ and oriT gene fragments were amplified from pDS132 using primers traJ-oriT+ and traJ-oriT-, and the amplified DNA fragments were purified using a PCR product recovery kit and digested with SpeI.

(3) The cre gene fragment was amplified from pDCW 109 using the primers Ptac-cre+ and Ptac-cre-, and the PCR product was recovered using a PCR product recovery kit.

(4) 1 mug of each of the cleavage products in the steps (1) and (2) was used to prepare a ligation system, and ligation was performed at 4℃overnight, and the ligation system was treated at 70℃for 10 minutes to inactivate the ligase.

(5) And (3) connecting the DNA fragment in the step (3) and the connection product in the step (4) by using a one-step cloning kit, converting to CC118 (lambda pir) competence, culturing for 24 hours at 37 ℃, picking a colony, verifying, extracting plasmid sequencing (the result is shown in figure 3), and obtaining the knocked-out plasmid with correct sequencing, wherein the plasmid is named as pOTC.

The plasmid pOTC with correct sequencing result is selected to be electrically transformed into S17-1 (lambda pir), and after colony verification is correct, the plasmid pOTC is cultured in LB liquid culture medium for 12 hours at 37 ℃, 800 mu L of bacterial liquid is taken and added into glycerol tube for seed protection at-70 ℃.

2. Construction of plasmid pOTC-SB

(1) Amplifying sacB fragments from pDS132 using primers sacb+ and sacB-, resulting in sacB fragments; the pOTC plasmid was enzymatically digested with BstZ17I, finally the sacB fragment was ligated with the linearized plasmid pOTC fragment using a one-step cloning kit and transformed into CC118 (λpir) for colony PCR validation and sequencing (results are shown in FIG. 3).

(2) The plasmid pOTC-SB with correct sequence is electrically transferred into S17-1 (lambda pir), and after colony verification is correct, the plasmid is cultured for 12 hours in LB liquid culture medium at 37 ℃, 800 mu L of bacterial liquid is taken and added into glycerol tube for seed protection at-70 ℃.

The construction flow of the pOTC-SB plasmid is shown in FIG. 2, and after the construction is completed, the enzyme digestion verification is carried out by using a fast-cutting enzyme BstZ17I, ecoRI, and the result shows that: the construction of the recombinant plasmid pOTC-SB containing the sacB gene was successful, and S17-1 (λpir) containing the plasmid pOTC-SB was obtained.

Example 2: construction of Vibrio parahaemolyticus Gene deletion mutant

Vibrio parahaemolyticus itself has a homologous recombination system. By utilizing this, suicide plasmids containing homology arms are integrated into the genome after entering cells, and the application is proved to be highly effective and successful in implementation by taking the genes with accession numbers VP_RS11975, VP_RS22195, VP_RS23020, VP_RS16800, VP_RS16465, VP_RS20840, VP_RS03765 and VP_RS11205 on NCBI of the sequences and rpoE with accession numbers VP_RS12550 on NCBI as examples.

The method comprises the following specific steps:

1. construction of Gene knockout Strain

(1) PCR amplification was performed using the upstream and downstream homology arm primers of VP_RS11975, VP_RS22195, VP_RS23020, VP_RS16800, VP_RS16465, VP_RS20840, VP_RS03765, VP_RS11205, rpoE in Table 1, to obtain the upstream and downstream homology arms of each gene, respectively.

(2) The loxL-Gm-loxR fragment was amplified from pWJW101 using primers Gm-and Gm+.

(3) And (3) integrating the upstream homology arm, loxL-Gm-loxR and the downstream homology arm of each gene in the step (1) and the step (2) by fusion PCR to obtain UP-loxL-Gm-loxR-DOWN containing the homology arm of each gene.

(4) Respectively carrying out enzyme digestion on the pDS132 plasmid and the UP-loxL-Gm-loxR-DOWN containing the homologous arm of each gene obtained in the step (3) by using XbaI or SalI (wherein the VP_RS23020 homologous arm is connected with the pDS132 plasmid subjected to enzyme tangential digestion by using a one-step cloning kit), so as to respectively obtain different connection products;

the ligation products were respectively subjected to overnight ligation at 4℃and then transformed into CC118 (λpir) and coated on LB plates containing gentamicin at a concentration of 30 mg/L; and (3) through colony PCR verification, picking up correct bacteria, culturing for 24 hours at 37 ℃, and respectively extracting plasmids. The plasmids were introduced into E.coli S17-1 (λpir) by electrotransformation, respectively, and plated on LB plates containing gentamicin at a concentration of 30mg/L.

(5) First engagement transduction:

e.coli S17-1 (λpir) and Vibrio parahaemolyticus ATCC33846 obtained in the step (4) and containing the LB plate are respectively cultivated to OD at 37 DEG C ₆₀₀ About 1.0, 1mL of each bacterial liquid was washed twice with LB liquid medium, 100. Mu.L of LB liquid medium was co-suspended, inoculated on LB plates, and cultured overnight. The lawn was suspended in 1mL of LB using a sterile cotton swab, and the suspension was spread on LB plates containing 10mg/L polymyxin B and 6mg/L chloramphenicol.

A first homologous recombination occurs at the first conjugation transduction, and single colonies on the LB plates are picked up for colony PCR and culture and extraction genome verification. The correct colony should be verified to have two bands, one length being the length of the homology arm and the gentamicin resistance gene on the plasmid and the other length being the length of the homology arm and the target gene on the genome.

(6) Sucrose screening:

inoculating the bacteria with correct verification in the step (5) into test tubes containing gentamicin with the concentration of 30mg/L and LB culture medium containing 10% (w/v) sucrose, culturing for 12 hours at 37 ℃, respectively taking 50 mu L of the bacteria coated on LB plates containing gentamicin with the concentration of 30mg/L and sucrose with the concentration of 10% (w/v), carrying out colony PCR and genome verification on single colonies, and at the moment, respectively carrying out second homologous recombination in different deletion mutant strains, removing pDS132 plasmid with target genes from the genome of Vibrio parahaemolyticus ATCC33846 to respectively obtain gene deletion mutant strains containing gentamicin resistance and lacking corresponding proteins.

(7) Second engagement transduction:

the S17-1 (λpir) containing the plasmid pOTC-SB obtained in step 2 of example 1 was ligated with the gene deletion mutant strain obtained in step (6) of the present method, respectively, according to the above-mentioned method of the ligation transduction test in step (5), to obtain the gene deletion mutant strain containing the plasmid pOTC-SB, respectively.

The single colonies were picked for verification, and there were three results:

the first is a band with only one length of upstream and downstream homology arms and celebration resistance genes;

the second is that only one band with the length of 100bp larger than that of the upstream and downstream homologous arms is adopted, namely the gentamicin resistance gene is removed;

the third is the appearance of both bands, i.e., colonies did not completely remove the pDS132 plasmid.

(8) For the first and third colonies, the colonies were picked and placed in LB liquid containing chloramphenicol resistance at a concentration of 30mg/L for culture at 37℃for 12 hours to allow more complete excision of Cre enzyme, and after the culture was completed, 50-200. Mu.L of the bacterial liquid was again applied to a chloramphenicol plate containing chloramphenicol at a concentration of 30mg/L for single colony verification.

For the second colony, the negative selection was performed by streaking on LB plates containing gentamicin at a concentration of 10 mg/L. Colonies which did not grow on the gentamicin plates were inoculated into a tube containing 10% (w/v) sucrose LB liquid medium overnight to remove the pOTC-SB plasmid, 50. Mu.L of the above-mentioned bacterial liquid from which the pOTC-SB plasmid was removed was plated on 10% (w/v) sucrose LB plates, and single colonies were streaked on chloramphenicol LB plates containing a concentration of 30mg/L for negative selection. The not-grown colonies were the correct knockout complete strains.

2. Expression of results:

(1) In the optimized knockout system, a gentamicin resistance gene is introduced between the upstream and downstream homology arms. To smoothly remove the gentamicin resistance gene after the completion of the knockout, a Cre/loxP system was introduced, and loxP sites were located at both ends of Yu Qingda resistance gene (FIG. 1). The Cre enzyme is able to recognize and cleave the loxP site. To allow the cre gene to enter the Vibrio parahaemolyticus ATCC33846 cells, a pOTC-SB plasmid was constructed (FIG. 2), and verification was performed after the plasmid construction was completed (FIG. 3).

(2) The application selects an rpoE gene verification optimized knockout system. After the second homologous recombination of the optimized knockout system, the length of the successfully knocked-out band should be the sum of the upstream and downstream homology arms and the celebrate gene, which is 2268bp. The unsuccessful band length is the sum of the upstream and downstream homology arms and the target gene, 1756bp.

From FIG. 4, it can be seen that among 10 single colonies randomly picked, 1 was knocked out successfully, and 9 plasmids were not completely removed.

And after the second homologous recombination of the original knockout system, the length of the successfully knocked-out band is the upstream and downstream homology arms and 1178bp. The unsuccessful band length is the sum of the upstream and downstream homology arms and the target gene, 1756bp. As can be seen from fig. 5, all 10 single colonies randomly picked returned to wild type. The optimized knockout system can screen out the thalli reverting to the wild type.

(3) The application removes the gentamicin resistance gene after the target gene is knocked out. As is clear from FIG. 6, after the plasmid pOTC-SB was introduced, the efficiency of Cre enzyme for removing the celebrating resistance gene between loxP sites was 20%, and 5 lines of 25 single colony plates were streaked to remove the celebrating resistance gene.

As can be seen from FIG. 7, the efficiency of removing pOTC-SB plasmid in 10% sucrose environment was 100% for 25 strains of the plasmid after streaking 25 single colony plates.

(4) The 8 genes were knocked out at one time using an optimized knockout system. The final knockdown strains were all successfully constructed (FIG. 8). The optimized knockout system is shown to have higher stability of results than the original one, reducing the repeated multiple passes of the second homologous recombination.

While the application has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the application as defined in the appended claims.

SEQUENCE LISTING

<110> university of Jiangnan

<120> method for constructing efficient knock-out plasmid for vibrio parahaemolyticus gene

<130> BAA220158A

<160> 3

<170> PatentIn version 3.3

<210> 1

<211> 4520

<212> DNA

<213> artificial sequence

<400> 1

tcgggtcggg tgaatcttgt tgatggcgat atggatgtgc aggttgtcgg tgtcgtgatg 60

cacggcactg acgcgctgat gctcggcgaa gccaagccca gcgcagatgc ggtcctcaat 120

cgcgcgcaac gtctccgcgt cgggcttctc tcccgcgcgg aagctaacca gcaggtgata 180

ggtcttgtcg gcctcggaac gggtgttgcc gtgctgggtc gccatcacct cggccatgac 240

agcgggcagg gtgtttgcct cgcagttcgt gacgcgcacg tgacccaggc gctcggtctt 300

gccttgctcg tcggtgatgt acttcaccag ctccgcgaag tcgctcttct tgatggagcg 360

catggggacg tgcttggcaa tcacgcgcac cccccggccg ttttagcggc taaaaaagtc 420

atggctctgc cctcgggcgg accacgccca tcatgacctt gccaagctcg tcctgcttct 480

cttcgatctt cgccagcagg gcgaggatcg tggcatcacc gaaccgcgcc gtgcgcgggt 540

cgtcggtgag ccagagtttc agcaggccgc ccaggcggcc caggtcgcca ttgatgcggg 600

ccagctcgcg gacgtgctca tagtccacga cgcccgtgat tttgtagccc tggccgacgg 660

ccagcaggta ggccgacagg ctcatgccgg ccgccgccgc cttttcctca atcgctcttc 720

gttcgtctgg aaggcagtac accttgatag gtgggctgcc cttcctggtt ggcttggttt 780

catcagccat ccgcttgccc tcatctgtta cgccggcggt agccggccag cctcgcagag 840

caggattccc gttgagcacc gccaggtgcg aataagggac agtgaagaag gaacacccgc 900

tcgcgggtgg gcctacttca cctatcctgc ccggctgacg ccgttggata caccaaggaa 960

agtggtacct gaacgacccc gaatattgga tcacagcgca gcaggtcgcc gcccgcgtag 1020

ctctcacccc ggccaccatt aaaaagtggg caaacgaggg aaaaatcacc gcatacaaga 1080

tcggcaagtc cgtccgattc aaagcatcag acgtagaccc gcgatgagct gttgacaatt 1140

aatcatcggc tcgtataatg tgtggaattg tgagcggata acaattagat ctggtaccct 1200

cgagagaagg agatatacca tgtccaattt actgaccgta caccaaaatt tgcctgcatt 1260

accggtcgat gcaacgagtg atgaggttcg caagaacctg atggacatgt tcagggatcg 1320

ccaggcgttt tctgagcata cctggaaaat gcttctgtcc gtttgccggt cgtgggcggc 1380

atggtgcaag ttgaataacc ggaaatggtt tcccgcagaa cctgaagatg ttcgcgatta 1440

tcttctatat cttcaggcgc gcggtctggc agtaaaaact atccagcaac atttgggcca 1500

gctaaacatg cttcatcgtc ggtccgggct gccacgacca agtgacagca atgctgtttc 1560

actggttatg cggcggatcc gaaaagaaaa cgttgatgcc ggtgaacgtg caaaacaggc 1620

tctagcgttc gaacgcactg atttcgacca ggttcgttca ctcatggaaa atagcgatcg 1680

ctgccaggat atacgtaatc tggcatttct ggggattgct tataacaccc tgttacgtat 1740

agccgaaatt gccaggatca gggttaaaga tatctcacgt actgacggtg ggagaatgtt 1800

aatccatatt ggcagaacga aaacgctggt tagcaccgca ggtgtagaga aggcacttag 1860

cctgggggta actaaactgg tcgagcgatg gatttccgtc tctggtgtag ctgatgatcc 1920

gaataactac ctgttttgcc gggtcagaaa aaatggtgtt gccgcgccat ctgccaccag 1980

ccagctatca actcgcgccc tggaagggat ttttgaagca actcatcgat tgatttacgg 2040

cgctaaggat gactctggtc agagatacct ggcctggtct ggacacagtg cccgtgtcgg 2100

agccgcgcga gatatggccc gcgctggagt ttcaataccg gagatcatgc aagctggtgg 2160

ctggaccaat gtaaatattg tcatgaacta tatccgtacc ctggatagtg aaacaggggc 2220

aatggtgcgc ctgctggaag atggcgatta gctgcagtgt ggaacaccta catctgtatt 2280

aacgaagcgc taaccgtttt tatcaggctc tgggaggcag aataaatgat catatcgtca 2340

attattacct ccacggggag agcctgagca aactggcctc aggcatttga gaagcacacg 2400

gtcacactgc ttccggtagt caataaaccg gtaaaccagc aatagacata agcggctatt 2460

taacgaccct gccctgaacc gacgaccggg tcgaatttgc tttcgaattt ctgccattca 2520

tccgcttatt atcacttatt caggcgtagc accaggcgtt taagggcacc aataactgcc 2580

ttaaaaaaat tacgccccgc cctgccactc atcgcagtac tgttgtaatt cattaagcat 2640

tctgccgaca tggaagccat cacagacggc atgatgaacc tgaatcgcca gcggcatcag 2700

caccttgtcg ccttgcgtat aatatttgcc catggtgaaa acgggggcga agaagttgtc 2760

catattggcc acgtttaaat caaaactggt gaaactcacc cagggattgg ctgagacgaa 2820

aaacatattc tcaataaacc ctttagggaa ataggccagg ttttcaccgt aacacgccac 2880

atcttgcgaa tatatgtgta gaaactgccg gaaatcgtcg tggtattcac tccagagcga 2940

tgaaaacgtt tcagtttgct catggaaaac ggtgtaacaa gggtgaacac tatcccatat 3000

caccagctca ccgtctttca ttgccatacg gaattccgga tgagcattca tcaggcgggc 3060

aagaatgtga ataaaggccg gataaaactt gtgcttattt ttctttacgg tctttaaaaa 3120

ggccgtaata tccagctgaa cggtctggtt ataggtacat tgagcaactg actgaaatgc 3180

ctcaaaatgt tctttacgat gccattggga tatatcaacg gtggtatatc cagtgatttt 3240

tttctccatt ttagcttcct tagctcctga aaatctcgat aactcaaaaa atacgcccgg 3300

tagtgatctt atttcattat ggtgaaagtt ggaacctctt acgtgccgat caacgtctca 3360

ttttcgccaa aagttggccc agggcttccc ggtatcaaca gggacaccag gatttattta 3420

ttctgcgaag tgatcttccg tcacaggtat ttattcggcg caaagtgcgt cgggtgatgc 3480

tgccaactta ctgatttagt gtatgatggt gtttttgagg tgctccagtg gcttctgttt 3540

ctatcagctg tccctcctgt tcagctactg acggggtggt gcgtaacggc aaaagcaccg 3600

ccggacatca gcgctagcgg agtgtatact ggcttactat gttggcactg atgagggtgt 3660

cagtgaagtg cttcatgtgg caggagaaaa aaggctgcac cggtgcgtca gcagaatatg 3720

tgatacagga tatattccgc ttcctcgctc actgactcgc tacgctcggt cgttcgactg 3780

cggcgagcgg aaatggctta cgaacggggc ggagatttcc tggaagatgc caggaagata 3840

cttaacaggg aagtgagagg gccgcggcaa agccgttttt ccataggctc cgcccccctg 3900

acaagcatca cgaaatctga cgctcaaatc agtggtggcg aaacccgaca ggactataaa 3960

gataccaggc gtttccccct ggcggctccc tcgtgcgctc tcctgttcct gcctttcggt 4020

ttaccggtgt cattccgctg ttatggccgc gtttgtctca ttccacgcct gacactcagt 4080

tccgggtagg cagttcgctc caagctggac tgtatgcacg aaccccccgt tcagtccgac 4140

cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggaaagaca tgcaaaagca 4200

ccactggcag cagccactgg taattgattt agaggagtta gtcttgaagt catgcgccgg 4260

ttaaggctaa actgaaagga caagttttgg tgactgcgct cctccaagcc agttacctcg 4320

gttcaaagag ttggtagctc agagaacctt cgaaaaaccg ccctgcaagg cggttttttc 4380

gttttcagag caagagatta cgcgcagacc aaaacgatct caagaagatc atcttattaa 4440

tcagataaaa tatttctaga tttcagtgca atttatctct tcaaatgtag cacctgaagt 4500

cagccccata cgatactagt 4520

<210> 2

<211> 1955

<212> DNA

<213> artificial sequence

<400> 2

cacctttatg ttgataagaa ataaaagaaa atgccaatag gatatcggca ttttcttttg 60

cgtttttatt tgttaactgt taattgtcct tgttcaagga tgctgtcttt gacaacagat 120

gttttcttgc ctttgatgtt cagcaggaag cttggcgcaa acgttgattg tttgtctgcg 180

tagaatcctc tgtttgtcat atagcttgta atcacgacat tgtttccttt cgcttgaggt 240

acagcgaagt gtgagtaagt aaaggttaca tcgttaggat caagatccat ttttaacaca 300

aggccagttt tgttcagcgg cttgtatggg ccagttaaag aattagaaac ataaccaagc 360

atgtaaatat cgttagacgt aatgccgtca atcgtcattt ttgatccgcg ggagtcagtg 420

aacaggtacc atttgccgtt cattttaaag acgttcgcgc gttcaatttc atctgttact 480

gtgttagatg caatcagcgg tttcatcact tttttcagtg tgtaatcatc gtttagctca 540

atcataccga gagcgccgtt tgctaactca gccgtgcgtt ttttatcgct ttgcagaagt 600

ttttgacttt cttgacggaa gaatgatgtg cttttgccat agtatgcttt gttaaataaa 660

gattcttcgc cttggtagcc atcttcagtt ccagtgtttg cttcaaatac taagtatttg 720

tggcctttat cttctacgta gtgaggatct ctcagcgtat ggttgtcgcc tgagctgtag 780

ttgccttcat cgatgaactg ctgtacattt tgatacgttt ttccgtcacc gtcaaagatt 840

gatttataat cctctacacc gttgatgttc aaagagctgt ctgatgctga tacgttaact 900

tgtgcagttg tcagtgtttg tttgccgtaa tgtttaccgg agaaatcagt gtagaataaa 960

cggatttttc cgtcagatgt aaatgtggct gaacctgacc attcttgtgt ttggtctttt 1020

aggatagaat catttgcatc gaatttgtcg ctgtctttaa agacgcggcc agcgtttttc 1080

cagctgtcaa tagaagtttc gccgactttt tgatagaaca tgtaaatcga tgtgtcatcc 1140

gcatttttag gatctccggc taatgcaaag acgatgtggt agccgtgata gtttgcgaca 1200

gtgccgtcag cgttttgtaa tggccagctg tcccaaacgt ccaggccttt tgcagaagag 1260

atatttttaa ttgtggacga atcgaattca ggaacttgat atttttcatt tttttgctgt 1320

tcagggattt gcagcatatc atggcgtgta atatgggaaa tgccgtatgt ttccttatat 1380

ggcttttggt tcgtttcttt cgcaaacgct tgagttgcgc ctcctgccag cagtgcggta 1440

gtaaaggtta atactgttgc ttgttttgca aactttttga tgttcatcgt tcatgtctcc 1500

ttttttatgt actgtgttag cggtctgctt cttccagccc tcctgtttga agatggcaag 1560

ttagttacgc acaataaaaa aagacctaaa atatgtaagg ggtgacgcca aagtatacac 1620

tttgcccttt acacatttta ggtcttgcct gctttatcag taacaaaccc gcgcgattta 1680

cttttcgacc tcattctatt agactctcgt ttggattgca actggtctat tttcctcttt 1740

tgtttgatag aaaatcataa aaggatttgc agactacggg cctaaagaac taaaaaatct 1800

atctgtttct tttcattctc tgtatttttt atagtttctg ttgcatgggc ataaagttgc 1860

ctttttaatc acaattcaga aaatatcata atatctcatt tcactaaata atagtgaacg 1920

gcaggtatat gtgatgggtt aaaaaggatc gatcc 1955

<210> 3

<211> 1092

<212> DNA

<213> artificial sequence

<400> 3

cgtaatacga ctcactatag ggcgaattgg gtaccgggcc ttaccgttcg tatagcatca 60

ttatacgaag ttatcgaacc ccacccgggg cattctgccg acatggaagc catcacaaac 120

ggcatgatga acctgaatcg ccagcggcat cagcaccttg tcgccttgcg tataatattt 180

gcccatgcat ggacgcacac cgtggaaacg gatgaaggca cgaacccagt tgacataagc 240

ctgttcggtt cgtaaactgt aatgcaagta gcgtatgcgc tcacgcaact ggtccagaac 300

cttgaccgaa cgcagcggtg gtaacggcgc agtggcggtt ttcatggctt gttatgactg 360

tttttttgta cagtctatgc ctcgggcatc caagcagcaa gcgcgttacg ccgtgggtcg 420

atgtttgatg ttatggagca gcaacgatgt tacgcagcag caacgatgtt acgcagcagg 480

gcagtcgccc taaaacaaag ttaggtggct caagtatggg catcattcgc acatgtaggc 540

tcggccctga ccaagtcaaa tccatgcggg ctgctcttga tcttttcggt cgtgagttcg 600

gagacgtagc cacctactcc caacatcagc cggactccga ttacctcggg aacttgctcc 660

gtagtaagac attcatcgcg cttgctgcct tcgaccaaga agcggttgtt ggcgctctcg 720

cggcttacgt tctgcccaag tttgagcagc cgcgtagtga gatctatatc tatgatctcg 780

cagtctccgg agagcaccgg aggcagggca ttgccaccgc gctcatcaat ctcctcaagc 840

atgaggccaa cgcgcttggt gcttatgtga tctacgtgca agcagattac ggtgacgatc 900

ccgcagtggc tctctataca aagttgggca tacgggaaga agtgatgcac tttgatatcg 960

acccaagtac cgccacctaa cccgggttgc ggcagcgtga atctcgatat aacttcgtat 1020

agcatacata tacgaacggt atagcggtgg agctccagct tttgttccct ttagtgaggg 1080

ttaattgcgc gc 1092

Claims (8)

1. A knockout plasmid pOTC, characterized in that the knockout plasmid pOTC comprises a repliconp15AResistance geneCmRTransfer elementoriTAndtraJpromoter Ptac、CreA gene; the nucleotide sequence of the knocked-out plasmid pOTC is shown as SEQ ID NO.1, and the knocked-out plasmid pOTC also containsSacBA gene; the saidSacBThe nucleotide sequence of the gene is shown as SEQ ID NO. 2.

2. A method for knocking out a gene of interest in vibrio parahaemolyticus, comprising the steps of:

(1) SynthesisloxL-Gm-loxRFragments of (a)loxL-Gm-loxRThe nucleotide sequence of the fragment is shown as SEQ ID NO. 3;

(2) Homologous arm of the target gene at the upstream and downstreamloxL-Gm-loxRThe fragment is integrated on a pDS132 plasmid to obtain a recombinant plasmid pDS132-1;

(3) Introducing the recombinant plasmid pDS132-1 into the competence of the escherichia coli to obtain recombinant escherichia coli/pDS 132-1;

(4) Recombinant escherichia coli/pDS 132-1 and vibrio parahaemolyticus are combined and transduced to obtain recombinant vibrio parahaemolyticus/pDS 132-1 containing recombinant plasmid pDS132-1, and the recombinant vibrio parahaemolyticus/pDS 132-1 is inoculated into a sucrose culture medium containing gentamicin for culture;

(5) Will containSacBThe knockout plasmid pOTC of claim 1 of the gene is introduced into escherichia coli competence to obtain recombinant escherichia coli/pOTC-SB; and (3) combining and transduction of recombinant escherichia coli/pOTC-SB and the recombinant vibrio parahaemolyticus cultured in the step (4), and inoculating the recombinant escherichia coli/pOTC-SB into a culture medium containing chloramphenicol for culture to obtain the vibrio parahaemolyticus from which the target gene is knocked out.

3. The method of claim 2, wherein in steps (3) and (5), the E.coli comprises CC118λ pir、S17-1 λpir。

4. The method of claim 3, wherein in step (4), the sucrose medium containing gentamicin is LB medium, and the sucrose is added in an amount of at least: 10% (w/v).

5. The method of claim 4, wherein the gentamicin is added in an amount of: 10-30 mg/L.

6. The method of claim 5, wherein the medium containing chloramphenicol is LB medium, and the chloramphenicol is added in an amount of: 6-30 mg/L.

7. The method according to any one of claims 2 to 6, wherein the gene of interest is any gene on the genome of Vibrio parahaemolyticus that does not affect its growth.

8. Use of the knockout plasmid of claim 1 for knocking out any gene on the genome of vibrio parahaemolyticus that does not affect its growth.