不同来源RNA聚合酶对霍乱弧菌分型噬菌体VP3启动子的作用

Effects of RNA Polymerase from Different Sources on the Predicted Promoters of Vibrio cholerae Typing Phage VP3

目的:研究不同来源的RNA聚合酶对预测的霍乱弧菌分型噬菌体VP3启动子的作用。方法:以含有预测的VP3启动子区的片段取代质粒pRL-null的T7启动子区,以海肾萤光素酶基因Rluc为报告基因,在霍乱弧菌N16961内检测霍乱弧菌RNA聚合酶对克隆的启动子区的作用;将上述重组质粒和表达VP3 RNA聚合酶的质粒共转化大肠杆菌JM109,检测大肠杆菌和VP3的RNA聚合酶对克隆的启动子区的作用。结果:N16961的RNA聚合酶不能识别并作用于启动子P1、P2、P5、P6、P10和P12,JM109的RNA聚合酶可能识别并作用于启动子P7和P11;只有P2、P7、P8、P9、P13、P16和P17在JM109内可以被克隆表达的VP3 RNA聚合酶识别转录。结论:宿主菌N16961与非宿主菌JM109的RNA聚合酶识别转录VP3启动子的能力不同,可能与噬菌体的宿主特异性有关;VP3的RNA聚合酶对大部分有活性的VP3启动子具有直接启动转录作用,但部分启动子可能需要VP3或宿主蛋白的辅助作用才能表现出更强的活性;VP3启动子对VP3 RNA聚合酶的特异性也不同,P1、P2和P12对VP3的RNA聚合酶具有高度特异性,P7和P11的特异性较弱。

Objective：To study the effects of RNA polymerase（RNAP） from different sources on the predicted promoters of Vibrio cholerae typing phage VP3.Methods：T7 promoter of plasmid pRL-null was replaced by the fragments containing the predicted VP3 promoters,and Rluc was used as the report gene.The resulting plasmids were transformed into V.cholerae N16961,and the effect of N16961 RNAP on VP3 promoters was observed.These plasmids were transformed into Escherichia coli JM109 as well as a plasmid expressing VP3 RNAP,and the effect of JM109 and VP3 RNAP on VP3 promoters was observed.Results：Promoters P1,P2,P5,P6,P10 and P12 couldn＇t be used by N16961 RNAP,promoters P7 and P11 could be used by JM109 RNAP.In JM109,promoters P2,P7,P8,P9,P13,P16 and P17 could be used by VP3 RNAP.Conclusion：RNAP from host N16961 and non-host JM109 have different capability in transcribing from VP3 promoters,probably due to the specificity of phage for host cells.VP3 RNAP can transcribe from most VP3 promoters directly,but part promoters may need the help of other VP3 or host proteins to show stronger activities.VP3 promoters showed different specificity for VP3 RNAP：P1,P2 and P12 showed high specificity,and P7 and P11 showed weak specificity.