一种基于温敏质粒的新型基因敲除方法

A Novel Temperatrue Sensitive Plasmid-based Method for Deletion of Chromosomal Genes

基于温敏型质粒而不用线性DNA的方法用于快速敲除沙门氏菌染色体上的目的基因。以伤寒沙门氏菌S.ty2基因组为模板扩增得到的ssaV基因的上下游同源臂,与两端带有FRT位点的卡那霉素抗性基因片段连接到温敏型质粒pHY304,共同构建同源重组载体;然后转化S.ty2,通过筛选得到带有抗性标记的重组菌。通过转入重组酶表达质粒pCP20,去除抗性标记,得到ssaV基因缺失的重组菌,并在DNA水平进行了鉴定。建立了一种改进的基于温敏质粒的沙门氏菌的基因敲除方法,此方法也值得在其他革兰氏阴性菌的基因敲除中尝试应用。

A temperature sensitive plasmid-based procedure without linear DNA was developed for a rapid deletion of chromosomal target genes in Salmonella.To construct a recombinant vector,homologous regions and kana cassette with two FRT sites were amplified using corresponding templates and cloned into Ts-plasmid pHY304.The produced shuttle vector was then transformed into S.ty2.Under the pressure of temperature and antibiotic,homologous recombination occurred between the vector and genome of the host bacterium and the recombinants were selected by agar media with kanamycin.To delete kan resistant cassette,plasmid pCP20 was introduced into the recombinant.The markerless mutant strains were detected by genome PCR.The ssaV gene was successfully deleted by the modified method.A novel Ts-plasmid-based method has been established,which could be used for deletion of gene in other Gram-negative bacteria.