Objective To construct a strain of Pseudomonas aeruginosa harboring mucA56 in order to investigate the impact on mucA function of a novel mutant mucA56. Methods MucA56 was subcloned into a suicide vector pEXlOOT to generate pEXmucA56 plasmid, which was subsequently transformed into E. Coli DH5α. After homologous recombination through co-culture of the transformed DH5α bacteria with a standard Pseudomonas aeruginosa strain PAO1 ,and selection using carbenicillin, Pseudomonas isolation agar plates and sucrose plates, a novel PAO1 strain with the new mucA mutant inserted to bacterial chromosome was generated. Results The pEXmucA56 plasmid and the homologous recombinant strain PAOmucA56 were validated by both enzyme restriction and PCR. Conclusion The successful construction of the PAOmucA56 strain facilitated further study on the mucA mutant.%目的 构建铜绿假单胞菌新的mucA基因突变菌株,为研究新突变的mucA基因功能提供实验菌株.方法 首先将含新突变的mucA基因同源片段与自杀质粒pEX100T相连接,构建质粒pEXmucA56,再将质粒pEXmucA56转化人大肠埃希菌DH5α,与铜绿假单胞菌标准菌株PAO1共培养进行同源重组,并用羧苄青霉素(Carbenicillin)平板、假单胞菌分离平板(Pseudomonas isolation agar,PIA)和蔗糖(sucrose)平板进行筛选,获得染色体上含新突变的mucA基因的PAO1菌株.结果 经酶切及聚合酶链反应(polyrnerase chain reaction,PCR)鉴定质粒pEXmucA56构建及转化成功,经PCR及测序分析证实同源重组菌株PAOmucA56构建成功.结论 成功构建了含新的mucA基因突变的铜绿假单胞菌菌株PAOmucA56,为研究新突变的mucA基因的功能奠定了基础.
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