以报告基因分析霍乱弧菌噬菌体VP1的预测启动子

VP1为感染并裂解霍乱弧菌的噬菌体,全基因组为环状双链DNA。通过测定该基因组序列,预测出15个可能的启动子区,利用报告基因质粒转化及全噬菌体共感染的策略分析了这些推测启动子在霍乱弧菌中的活性,将预测的启动子区分别克隆到启动子探测lacZ融合质粒载体pRS1274,在转化于大肠埃希氏菌受体菌株JM109中时,所有克隆子均呈现兰斑。同时将质粒电击到缺失了lacZ基因的霍乱弧菌菌株7743△Z,然后用噬菌体VP1感染转化菌株。在转化成功的13个含预测启动子片段的重组质粒中,通过检测β_半乳糖苷酶活性表达随感染后时间的变化,提示P17为早期启动子,P2、P3、P9等为中期启动子,P18为晚期启动子。

Functional analysis of promoters of Vibrio cholerea typing phage VP1 with reporter system

Phage VP1 infects and lyses Vibrio cholerae. The VP1 genome is a circular double-strand DNA and its size is 32176 base pairs. Analysis of the sequence of the VP1 genome revealed the presence of 15 putative promoter sequence. The activities of these putative promoters in V. cholerae were assayed by transformation of reporter gene plasmid and phage infection together. Promoter regions were ligated into pRS1274/BamH I/EcoR I. Then transformed into E. coli JM109 and all of clone display blue. The recombinant plasmids were transformed into V. cholerae 7743 deltaZ by electroporation, then bacteriophage VP1 infect transformant. The time-course expressing lacZ gene and detecting change of beta-galactosidase enzyme activity in V. cholerae transformants at latent period, indicated P17 probably is a early promoter; P2 and P3 and P9 etc are medium-term promoters; P18 is a late promoter.