类鼻疽伯克霍尔德菌ΔrelA缺失株的构建及其生物学特性比较

Construction of Burkholderia pseudomalleiΔrel A mutant and comparison of their biological characteristics

目的:敲除类鼻疽伯克霍尔德菌严谨反应信号分子(p)ppGpp合成相关基因relA,探讨其对细菌的生长、泳动和生物被膜形成的生物学功能的影响。方法:将甲氧苄啶(trimethoprim resistance,TPR)抗性片段通过BglⅡ酶切位点经酶切酶连的方法对含SacB蔗糖致死基因的pK18mobSacB自杀性质粒进行改造,从而获得具有甲氧苄啶抗性的质粒(TPR-pK18mobSacB);通过同源重组基因的敲除方法对类鼻疽伯克霍尔德菌HNBP001的relA进行敲除,获得relA缺失菌株并测定比较其缺失前后生长、泳动、生物被膜的表型变化。结果:成功获得relA缺失菌株;ΔrelA的生长速度、泳动性及生物被膜形成均下降。结论:TPRpK18mobSacB改造质粒提高HNBP001菌株的敲除效率,可广泛应用于类鼻疽伯克霍尔德菌的基因敲除,为实验室从分子水平研究类鼻疽伯克霍尔德菌基因功能提供便利;relA基因的缺失可抑制HNBP001的生长、泳动和生物被膜形成。

Objective:The relA gene,a gene associated with the synthesis of rigorous response signaling molecule(p)ppGpp in Burkholderia pseudomallei(B.pseudomallei),was knocked out and its effects on the biological functions of bacterial growth,motility and biofilm formation were investigated.Methods:The plasmids with trimethoprim resistance(TPR)were modified by enzyme digestion and enzyme ligation method.The TPR fragment was linked to the suicide plasmid pK18mobSacB containing SacB sucrose killing gene by BglⅡenzyme digestion site,and trimethoprim resistance was obtained Plasmid(TPRpK18mobSacB);The relA gene of B.pseudomallei HNBP001 was knocked out by homologous recombinant gene knockout method,and the relA mutant was obtained.The growth,motility and biofilm phenotypes of B.pseudomallei HNBP001 were compared before and after the deletion of relA gene.Results:The relA mutant was successfully constructed.The growth rate,motility and biofilm formation ofΔrelA decreased.Conclusion:The modified plasmid TPR-pK18mobSACB can improve the knockout efficiency of HNBP001 strain,which can be widely used in the gene knockout of B.pseudomallei,and provide convenience for the laboratory to study the gene function of B.pseudomallei at the molecular level;The mutant of the relA gene inhibits the growth,motility and biofilm formation of HNBP001.