沙门菌Hfq依赖型小RNA GcvB靶基因trpE的鉴定  

Identification of Salmonella Hfq-dependent small RNA GcvB target gene trpE

作　　者：潘永 杨阳 李晨 刘丽娟 杨琦[1,2,5] PAN Yong;YANG Yang;LI Chen;LIU Li-juan;YANG Qi(College of Animal Science,Guizhou University,Guiyang 550025,China;Institute of Animal Diseases,Guizhou University,Guiyang 550025,China;Guizhou Province Livestock and Poultry Resource Genetic Management Station,Guiyang 550025,China;Duyun City Animal Disease Prevention and Control Center,Guizhou 558000,China;Guizhou Key Laboratory of Animal Diseases and Veterinary Public Health,Guiyang 550025,China)

机构地区：[1]贵州大学动物科学学院,贵州贵阳550025 [2]贵州大学动物疫病研究所,贵州贵阳550025 [3]贵州省畜禽资源遗传管理站,贵州贵阳550025 [4]都匀市动物疫病预防控制中心,贵州都匀558000 [5]贵州省动物疫病与兽医公共卫生重点实验室,贵州贵阳550025

出　　处：《中国兽医学报》2020年第9期1777-1782,共6页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(31760740);国家自然科学基金资助项目(31602065);贵州省科技厅联合资金项目贵州大学2017年度学术新苗培养及创新探索专项资助项目(黔科合LH字[2017]7263,黔科合平台人才[2017]5788);贵州省科技厅基金资助项目(黔科合基础[2016]1047)。

摘　　要：trpE基因在鼠伤寒沙门菌中编码邻氨基苯甲酸合酶Ⅰ并参与氨基酸生物合成和代谢。为鉴定鼠伤寒沙门菌Hfq依赖型小RNA GcvB所调控的基因trpE,本研究基于前期鼠伤寒沙门菌Hfq基因敲除株及gcvB基因敲除株转录组分析基础上,利用Red同源重组系统构建trpE基因lac融合菌株,并利用P22噬菌体转导技术构建gcvB和Hfq基因单缺失和双缺失菌株,通过β半乳糖苷酶试验检测trpE基因在不同情况下蛋白表达活性,结合生物信息学系统TargetRNA2预测GcvB R1区与trpE mRNA的作用位点。结果表明,与野生型菌株相比,gcvB基因敲除导致trpE基因蛋白表达量增加1.2倍,Hfq基因敲除后表达量增加1.1倍,而双敲除增加量仅为0.5倍。Target RNA2预测trpE mRNA与GcvB R1区可形成8个连续或18个不连续的碱基互补。以上结果表明trpE基因受GcvB和Hfq调控,且极大可能受GcvB直接调控。本研究为鼠伤寒沙门菌GcvB的作用机理研究奠定理论基础,丰富了GcvB的靶基因数目。In order to identify the gene trpE regulated by Salmonella typhimurium Hfq-dependent small RNA GcvB,based on the transcriptome analysis of the pre-S.typhimurium Hfq knockout strain and gcvB knockout strain,first using Red homologous recombination,lac fusion strain of trpE gene was constructed,and the single deletion and double deletion strains of gcvB and Hfq genes were constructed by P22 phage transduction technique,and then the protein of trpE gene under different conditions was detected byβ-galactosidase assay.The expression activity was finally combined with the bioinformatics system Target RNA2 analysis to predict the site of action of the GcvB R1 region and trpE mRNA.The results showed that knockdown of the gcvB gene resulted in a 1.2-fold increase in the protein expression of the trpE gene compared to the wild-type strain,1.1-fold after Hfq knockout,and only 0.5-fold in double knockout.Target RNA2 predicts that trpE mRNA can form 8 consecutive or 18 discrete base complements to the GcvB R1 region.These results indicate that the trpE gene is regulated by GcvB and Hfq and is highly likely to be directly regulated by GcvB.This study laid a theoretical foundation for the study of the mechanism of action of Salmonella typhimurium GcvB,enriching the number of target genes of GcvB.

关 键 词：鼠伤寒沙门菌 小RNA HFQ β半乳糖苷酶试验 trpE基因 

分 类 号：S855.1[农业科学—临床兽医学] R535[农业科学—兽医学]