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【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。
【Objective】 The aim of this study was to screen the genes that regulate Vibrio cholerae C6706-mediated expression of LphR family protein AphB. 【Method】 The C6706-aphB promoter region of V. cholerae Elto type strain was cloned into two reporter plasmids pBBRLux and pKP302 and introduced into Vibrio cholerae C6706- as a starting strain. A random mutagenesis library of LZV630-302 transposon was constructed by conjugating the starting strain with transposon pSC123. The expression of aphB promoter was detected by measuring the chemiluminescence intensity, and the mutants affected by aphB expression were screened. Transposon insertion sites were detected by random PCR and sequenced for gene analysis. [Results] Two mutant strains T1 and T2 that affected aphB expression (all leading to decrease) were obtained from 7 transposon libraries (about 40,000 mutant strains). Sequence alignment revealed that the T1 transposon was inserted in the vc1585 reading frame and the T2 transposon inserted at 7 bp from the end of the vc1602 gene. 【Conclusion】 The mutants with aphB expression change, genes vc1585 and vc1602, may directly or indirectly affect the expression of aphB, which lays the foundation for further study on the influencing factors of aphB expression regulation.