金黄色葡萄球菌一氧化氮合酶基因(nos)缺失突变株的构建

Objective: In order to investigate the function of nitric oxide synthase gene (nos) in prokaryote, the nos deletion mutant of S. aureus strain was constructed by homologous recombination. Methods: The upstream and downstream sequences of nos were generated by PCR using chromosomal DNA of S. aureus RN6390 as template. The homologous recombination vector pMAD△nos was constructed based on shuttle vector pMAD (carrying the screening markers, the temperature-sensitive replication origin, erythromycin resistance gene (erm), and β-lactase gene(bgaB)). S. aureus strain RN4220, a restriction deficient strain, was used as the initial recipient for transformation and the modified vector was then electroporated into S. aureus RN6390. The strain was incubated alternately at 30℃ and 42℃ and the nos deletion mutants were selected by antibiotic resistance and β-lactase activity. Results: Results from genome PCR, real-time quantitative PCR and direct sequencing indicates that the nos gene of the screened mutants was deleted from chromosomal DNA of S. aureus RN6390. Conclusions: The successfully constructed nos deletion mutant of S. aureus offered a useful tool to investigating the function of nos gene in S. aureus.