Universal gene-knockout suicide vector for vibrios and application thereof

Abstract

The invention discloses a universal gene-knockout suicide vector for vibrios and a construction method theroef and provides an application thereof in gene knockout of the vibrios. The universal gene-knockout suicide vector pLP12 is a ring-shaped vector and comprises a PBAD promoter, a repressor protein gene araC, an RP4 transferring initiation site, a chlorampenicol resistant gene, an R6K duplicating initiation site, a multiple-cloning-site area and a lethal gene vmi480; the multiple-cloning-site area at least contains two AhdI restriction enzyme digestion sites; the suicide vector pLP12 is subject to AhdI restriction enzyme digestion to form linearized suicide vector pLP12T. The universal gene-knockout suicide vector adopts entirely-new reverse selection genes vmi480 and is used for replacing the common sacB gene. Foreign fragments carried by the pLP12T are transferred to vibrio cells to be mutated by a jointing mode, under the pressure of antibiotics and reverse selection of products of lethal gene vmi480, first-time homologous recombination and second-time homologous recombination are carried out on the vibrios successively, and finally the mutant strain with deletion of target genes is generated.

Description

Technical field: [0001] The invention belongs to the field of microbial genetic manipulation, and in particular relates to a universal gene knockout suicide vector for Vibrio and its application. Background technique: [0002] Vibrio is the most common heterotrophic bacteria in the marine environment. At least 12 species of Vibrio can cause human disease, among which Vibrio cholerae, Vibrio vulnificus, and Vibrio parahaemolyticus are the most serious hazards to human health. In addition, many types of Vibrio are also pathogens of aquatic economic animals, and the vibriosis caused by them leads to a large amount of economic losses in aquaculture. Although some key pathogenic genes of Vibrio cholerae and Vibrio parahaemolyticus have been discovered very early, scientists' understanding of the pathogenic mechanism of Vibrio still lags behind the pathogenic bacteria of Enterobacteriaceae. The lack of mature and convenient genetic manipulation technology is the main obstacle for...

AI Technical Summary

Problems solved by technology

The gene knockout technology that can be successfully used for homologous recombination in bacteria such as Escherichia coli has some obvious defects in Vibrio gene knockout, including: (1) lack of versatility, limited to gene deletion in a certain Vibrio; (2) sacB is widely used as a lethal gene for suicide vectors, but the lethal effect induced by sacB is very weak in Vibrio, resulting in a very low proportion of correct deletion mutant clones in the reverse selection plate, resulting in a large number of deletion mutations Screening work; (3) It is time-consuming and labor-intensive to perform double enzyme digestion on the overlapping PCR product of the target fragment and the suicide plasmid; (4) The suicide plasmid itself is relatively large and contains the mobI mobile gene, which not only results in low efficiency of plasmid conjugation transfer, and has the potential to cause non-specific integration

(5) The recipient bacteria must have different resistance markers from the donor bacteria and suicide vectors, which limits the application of conventional techniques

Therefore, when used for gene knockout in Vibrio, the existing suicide vectors show obvious limitations, and it is urgent to develop new universal suicide vectors to overcome the above defects and realize the gene knockout technology based on the bacterial recBCD homologous recombination system Wide application in Vibrio

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