Construction of a vscC in-frame deletion mutant of Vibrio alginolyticus.

[Objective] The purpose of this study was to construct a vscC in-frame deletion mutant of Vibrio alginolyticus with no antibiotic resistance marker.[Method] The first vscC mutant molecules in vitro were generated by SOE-PCR and then ligated to a suicide vector pDM4 to construct a suicide recombinant vector pDM4-△vscC.To clone and replicate the recombinant vector,it was transformed to E.coli SY327 strain,and then positive clones were selected and proved by PCR analysis.After that,the pDM4-△vscC DNA was extracted in large numbers and transformed to the E.coli S17-1 strain that acted as a donor in bacterial conjugation using the heat shock method.The recombinant E.coli S17-1 strains then transferred the pDM4-△vscC to V.alginolyticus ZJ51-O by conjugation method;transconjugants were screened and selected sequentially using antibiotic selection strategy and sucrose based counter-selection system to find the suspected mutants wanted.Finally the suspected mutants were identified by PCR and confirmed by sequencing analysis.[Result] ZJ51-O△vscC was successfully constructed.[Conclusion] This study laid a foundation for further research on the function of vscC gene and molecular mechanism of type III secretion system in V.alginolyticus.Simultaneously,by the effective method other unknown functional genes in V.alginolyticus genome would be researched.