[ Objective] We identified genes that regulate the expression of aphB, the gene encoding a key virulence regulator in Vibrio cholerae 01 El Tor C6706 ~. [Methods] We constructed a transposon library in V. Cholerae C6706 ~ strain containing a PaphB-luxCDABE and PaphB-lacZ transcriptional reporter plasmids. Using a chemiluminescence imager system, we rapidly detected aphB promoter expression level at a large scale. We then sequenced the transposon insertion sites by arbitrary PCR and sequencing analysis. [Results] We obtained two candidate mutants Tl and T2 which displayed reduced aphB expression from approximately 40,000 transposon insertion mutants. Sequencing analysis shows that Tn inserted in vcl585 reading frame in the Tl mutant and Tn inserted in the end of coding sequence of vcl602 in the T2 mutant. [Conclusion] By using a genetic screen, we identified two potential genes that may involve in regulation of the expression of the key virulence regulator AphB. This study sheds light on our further investigation to fully understand V. Cholerae virulence gene regulatory cascades.%[目的]筛选霍乱弧菌C6706-中调控LysR家族蛋白AphB表达的基因.[方法]将霍乱弧菌埃尔托型菌株C6706-aphB启动子区克隆到2个报告质粒pBBRLux和pKP302上,并将其导入霍乱弧菌C6706-中,以此作为出发菌株.利用出发菌株与转座子pSC123接合构建LZV630-302转座子随机突变文库,通过测定化学发光强度检测aphB启动子的表达水平,筛选aphB表达受影响的突变株.利用随机PCR方法检测转座子插入位点,并测序比对分析基因.[结果]从7个转座子库中(共约4万个突变株)得到能影响aphB表达(均导致下降)的2株突变株T1和T2.测序比对发现T1中转座子插入在vc1585读码框内,T2中转座子插入在距vc1602基因末端7 bp处.[结论]获得aphB表达改变的突变株,基因vc1585和vc1602可能直接或间接影响aphB表达,为进一步研究aphB表达调控影响因素奠定了基础.
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