【摘要】 【目的】为了研究铜绿假单胞菌全局调控因子RsmA对两个吩嗪(Phenazine)合成基因簇phz1和phz2的调控方式与机制。【方法】采用基因缺失和抗性基因(gentamycin resistance cassette,aacC1)插入相结合的策略构建了rsmA基因缺失突变株PA-RG;通过构建互补表达载体和过表达载体,进一步确认RsmA对绿脓菌素的调控作用;采用电转化方法将构建的翻译融合表达载体pMEZ1(phz1’-’lacZ)和pMEZ2(phz2’-’lacZ)分别导入铜绿假单胞菌突变株PA-RG和野生株PAO1,采用Miller法测定融合β-半乳糖苷酶活性。【结果】在GA培养基中,互补分析和过表达分析表明,RsmA抑制绿脓菌素的合成。此外,pMEZ1在突变株PA-RG中的表达增强,为野生株的2-3倍;而pMEZ2在突变株PA-RG中的表达降低,野生株是突变株的2倍。【结论】由此初步判定,铜绿假单胞菌全局调控因子RsmA对两个不同吩嗪合成基因簇的调控作用具有特异性,在一定程度上RsmA负调控phz1,正调控phz2。更多还原

【Abstract】 In many Pseudomonas,RsmA mediates the production of a set of secondary metabolites or virulence factors.[Objective] Our aim is to evaluate the function and regulation of the rsmA gene on two phenazine-producing operons in Pseudomonas aeruginosa PAO1.[Methods] We first cloned the upstream and downstream fragments of the rsmA gene from the chromosomal DNA.With the insertion of gentamycin resistance cassette(aacC1),the deletion mutant PA-RG was created and verified with PCR.To complement and overexpress the rsmA gene,pME10R and pME32R were also constructed.By constructing the translational fusion plasmids phz1′-′lacZ pMEZ1and phz2′-′lacZ pMEZ2,we introduced them into the wild type strain PAO1 and the mutant PA-RG,respectively.Activities of beta-galactosidase were determined with Miller method.[Results] In glycerol-alanine medium,overexpression of the rsmA gene results in dramatical decrease of pyocyanin production in PA-RG and PAO1 strain.In addition,beta-galactosidase activity of phz1′-′lacZ in the mutant PA-RG was much higher than that in the wild type strain.However,beta-galactosidase activity of phz2′-′lacZ in the wild type strain was 2fold more than that in the mutant PA-RG.[Conclusion] The regulation mediated by RsmA on two phenazine loci is specific and differential.更多还原