霍乱弧菌中调控aphB 的基因筛选及其功能

Isolation and function of genes regulating aphB expression in Vibrio cholerae

摘要:

摘要:【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。

Abstract:

Abstract:［Objective］We identified genes that regulate the expression of aphB，the gene encoding a key virulence regulator in Vibrio cholerae O1 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 T1 and T2 which displayed reduced aphB expression from approximately 40，000 transposon insertion mutants． Sequencing analysis shows that Tn inserted in vc1585 reading frame in the T1 mutant and Tn inserted in the end of coding sequence of vc1602 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.