文献类型：期刊文献

中文题名：基于转录机器全局扰动筛查迟钝爱德华菌毒力相关基因

英文题名：Screening of virulence-associated genes of Edwardsiella tarda based on disturbance of transcription machinery

作者：王晓波[1];叶江[1];宋姗姗[1];王克平[1];张惠展[1]

机构：[1]华东理工大学生物反应器工程国家重点实验室,上海200237

年份：2013

卷号：26

期号：1

起止页码：36

中文期刊名：中国生物制品学杂志

外文期刊名：Chinese Journal of Biologicals

收录：CSTPCD;;Scopus;CSCD:【CSCD_E2013_2014】；

语种：中文

中文关键词：转录扰动;爱德华菌属,迟钝;rpoE基因;过量表达

外文关键词：Transcriptional perturbation; Edwardsiella; tarda; rpoE gene; Over-expression

摘要：目的通过转录机器(RNA聚合酶)σE因子过量表达实现全局转录扰动,筛查迟钝爱德华菌(Edwardsiellatarda,E.tarda)毒力相关基因。方法从E.tarda EIB202中PCR扩增RNA聚合酶σE因子编码基因rpoE,连接到载体pACYC184上,构建rpoE基因过量表达菌株;在克隆rpoE基因时引入突变,使该基因表达失活,构建移码突变失活菌株。感染模式生物斑马鱼,检测各菌株半数致死剂量(LD50),RT-PCR法检测各菌株rpoE及其下游调控基因degP、fkpA、plsB、lpxD、yfiO mRNA的转录水平。结果重组质粒pACYC184-rpoE经双酶切及测序证实构建正确;在102、103、104和105CFU/g注射浓度下,空载体转染菌株和rpoE基因过量表达菌株的毒力差异无统计学意义(P>0.05);与空载体转染菌株相比,rpoE基因过量表达菌株rpoE基因mRNA的转录水平上调9.5倍;与rpoE基因移码突变失活菌株相比,rpoE基因过量表达菌株rpoE基因下游调控基因degP、fkpA、plsB、lpxD、yfiO分别上调6.9、2.64、2.85、2.38、1.34倍。结论成功构建了E.tarda rpoE基因过量表达菌株以及移码突变失活菌株,初步确定degP、fkpA、plsB、lpxD、yfiO 5个基因与E.tarda毒力相关。
Objective The whole transcription process was disturbed by over-expression of σE factor of transcription machinery（RNA polymerase） so as to screen the virulence-associated genes of Edwardsiella tarda.Methods The rpoE gene encoding the σE factor of RNA polymerase was amplified from E.tarda EIB202 by PCR and inserted into vector pACYC184,based on which a recombinant E.coli strain for over-expression of rpoE gene was established.Mutation was introduced during cloning of rpoE gene to inactivate the gene expression,based on which a recombinant E.coli strain with shift mutation was established.The LD50 of various strains were determined by infection of zebra fish model,while the transcription levels of rpoE,degP,fkpA,plsB,lpxD and yfiO mRNAs by RT-PCR.Results Restriction analysis and sequencing proved that recombinant plasmid pACYC184-rpoE was constructed correctly.The virulences of recombinant E.coli strain for over-expression of rpoE gene,at concentrations of 102,103,104 and 105 CFU / g,showed no significant difference with those of E.coli transfected with empty vector（P 0.05）.The transcription level of rpoE mRNA in the recombinant E.coli strain for over-expression was 9.5 times higher than that in the E.coli strain transfected with empty vector.However,as compared with those in recombinant E.coli strain with shift mutation,the transcription levels of degP,fkpA,plsB,lpxD and yfiO mRNAs increased by 6.9,2.64,2.85,2.38 and 1.34 folds respectively.Conclusion Recombinant E.coli strain for over-expression of rpoE gene of E.tarda as well as that with shift mutation were successfully established,and degP,fkpA,plsB,lpxD and yfiO was preliminarily identified as virulence-associated genes of E.tarda.