OxyR蛋白对不同应激条件下伤寒沙门菌生存能力的影响

Influence of OxyR on the survival ability of Salmonella enterica serovar Typhi in stress conditions

目的:研究OxyR蛋白对伤寒沙门菌(Salmonella enterica serovar Typhi,S.Typhi)在应激条件下生存能力的影响。方法:用λ-Red系统制备S.Typhi oxyR基因缺陷变异株;通过重组质粒pBAD/gⅢ将oxyR基因和pBAD/gⅢ空质粒分别导入oxyR基因缺陷变异株;构建oxyR缺陷回补株和oxyR缺陷回补空质粒株;制作生长曲线,观察野生株、oxyR缺陷株、oxyR缺陷回补株及oxyR缺陷回补空质粒株在不同应激条件下的生长状况;实时荧光定量PCR(qRT-PCR)分析OxyR对S.Typhi氧化调节相关基因表达的影响。结果:成功制备S.Typhi oxyR基因缺陷变异株、oxyR基因缺陷回补株和空质粒对照株;生长能力分析结果表明,与野生株相比,oxyR缺陷株在氧应激时生长缓慢(P<0.05),oxyR缺陷回补株的生长能力得到恢复;qRT-PCR结果显示,在氧应激时OxyR蛋白可以抑制hemH基因的表达,活化uxuA基因的表达。结论:OxyR蛋白参与S.Typhi对氧应激的调节,为进一步研究S.Typhi的致病性奠定了基础。

Objective： To investigate the influence of OxyR on the survival of Salmonella enterica serovar Typhi（S.Typhi） under stress conditions.Methods： The oxyR deleted mutant of S.Typhi was prepared by the λ-Red recombination system;the oxyR complementary strain and the control strain were constructed by transferring the recombinant plasmid pBAD-oxyR and the empty vector pBAD/g Ⅲ into the oxyR deleted mutant,respectively.Growth curves of the wild type,oxyR deleted mutant,oxyR complementary and control strains were performed to observe the growth of S.Typhi in different stress conditions.Quantitative real-time polymerase chain reaction（qRT-PCR） analysis was used to examine the influence of OxyR on oxidative stress-related genes in S.Typhi.Results： The oxyR deleted mutant,the oxyR complementary and the control strain of S.Typhi were constructed successfully.The results of growth curves revealed that under oxidative stress,the growth of oxyR deleted mutant was significantly slower compared with the wild type（P＆lt;0.05）;and the ability was restored in the oxyR complementary strain.qRT-PCR results demonstrated OxyR could repress the expression of hemH,but activate uxuA,which were regulated by OxyR.Conclusion： OxyR was involved in the resistance of S.Typhi to oxidative stress,which laid a foundation for further research on S.Typhi virulence.