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Method for traceless knockout of ralstonia solanacearum gene and application thereof

A technology of traceless knockout and R. solanacearum, which is applied in the field of genetic engineering, can solve the problems of inconvenient operation and knockout failure, and achieve the effect of simple operation, less operation steps and high resistance screening efficiency

Active Publication Date: 2022-01-14
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this method can seamlessly knock out the genes of Helicobacter pylori, it needs to construct a knockout template plasmid, which is not easy to operate, and the knockout method of different bacteria may not be applicable, and the knockout may fail for some reasons. There is no report on the method of knocking out R. solanacearum gene without trace

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  • Method for traceless knockout of ralstonia solanacearum gene and application thereof
  • Method for traceless knockout of ralstonia solanacearum gene and application thereof
  • Method for traceless knockout of ralstonia solanacearum gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 The traceless knockout of R. solanacearum gene phcA

[0038]Ralstonia solanacearum has a variety of virulence factors, including exopolysaccharide (EPS), cell wall degrading enzyme (CWDE), secretion system and so on. Existing studies (Genin et al., 2012) have elucidated the complex regulatory mechanism of R. solanacearum. Among them, PhcA, the product of the gene phcA, is located in the center of the complex regulatory network and is a transcriptional regulator of the LysR family, which directly or indirectly regulates the production of EPS, CWDE and other virulence factors in a population density-dependent manner.

[0039] The gene accession number of the phcA gene of Ralstonia solanacearum is AC251_03705, its length is 1044bp, and it encodes a key regulatory factor of the phc QS system. Previous studies have shown (Wang Dechen, 2016) that the deletion of the R. solanacearum gene phcA will reduce its exopolysaccharide, which is a relatively intuitive phenoty...

Embodiment 2

[0093] Example 2 The traceless knockout of R. solanacearum EP1 gene epsB

[0094] The present invention adopts the same method to knock out the R. solanacearum exopolysaccharide gene epsB (the gene accession number is AC251_23630).

[0095] According to the sequence of the epsB gene, the primers for amplification of the upstream and downstream homology arms, the fragments of the upstream and downstream homology arms of R. solanacearum gene epsB, the gentamycin resistance gene fragment and the electrophoresis results of the fusion fragment of the three are as follows figure 2 As shown, 1 is the upstream homology arm fragment of epsB, 2 is the gentamicin resistance gene fragment with FRT sites at both ends, and 3 is the upstream homology arm and downstream homology arm fragment of epsB, composed of figure 2 It can be seen that the fusion fragment of the gene epsB was successfully obtained. The screening results of epsB gene knockout mutants with FRT sites and gentamicin resis...

Embodiment 3

[0098] The knockout of the Ralstonia solanacearum gene of embodiment 3 different hosts

[0099] In addition, the present invention adopts the method for seamlessly knocking out R. solanacearum genes described in Example 1 to carry out seamless knockout of different genes in R. solanacearum of different plant hosts, and detects the obtained scarless knockout mutants colony morphology. The present invention respectively knocks out the phcA gene in the R. solanacearum EP1 strain whose host is eggplant, the phcB gene in the R. solanacearum GMI1000 strain whose host is tomato, and the phcB gene in the R. solanacearum NS25 strain whose host is casuarina, The colony surface morphology of the three knockout mutants obtained is as follows: Figure 8 As shown, the left is the mutant obtained by knockout by the traditional method, and the right is the knockout mutant obtained by the scarless knockout method of the present invention. Depend on Figure 8 As can be seen, the mutant strai...

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Abstract

The invention discloses a method for traceless knockout of ralstonia solanacearum gene and application thereof. The method comprises the following steps: constructing a fusion fragment of a target gene to be knocked out, transferring the fusion fragment into a ralstonia solanacearum competent cell to obtain a target gene knockout mutant with an FRT site and antibiotic resistance, then constructing an auxiliary plasmid, transferring the auxiliary plasmid into the knockout mutant, and carrying out subculture to eliminate the antibiotic resistance, and the traceless knockout of the ralstonia solanacearum gene is realized. Compared with a traditional amphiphilic or triparental hybridization method, the gene knockout method provided by the invention has the advantages of being easy and convenient to operate, high in accuracy rate and the like, recombinant plasmids of target genes do not need to be constructed in the knockout process, and the defects of low efficiency of amphiphilic or triparental mating transformation, incomplete suicide of sucrose suicide plasmids, retention of plasmid DNA in mutant genomes and the like are also avoided. The traceless gene knockout method can also be used for knocking out different genes in ralstonia solanacearum of different hosts, has a wide application range.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering. More specifically, it relates to a method for knocking out genes of Ralstonia solanacearum and its application. Background technique [0002] Gene knockout is a technology to inactivate or delete the target gene through a certain way, and it is the most effective and direct technical method to study and analyze gene function. Gene knockout in the general sense mainly uses the principle of DNA homologous recombination to construct a homologous arm recombination plasmid of the target gene with the help of the sucrose suicide plasmid pK18, and then enters the recipient bacteria through triparental mating or electric shock to perform homologous recombination. Exchange, and finally use sucrose to remove the suicide plasmid, so as to achieve the purpose of knocking out the target gene. However, the traditional gene knockout method has the disadvantages of cumbersome experimental steps for...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/74C12N15/31C12R1/01
CPCC07K14/195C12N15/74Y02A50/30
Inventor 张炼辉闫金利廖立胜王小晴
Owner SOUTH CHINA AGRI UNIV
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