大肠杆菌yigP基因启动子的定位

Positioning of Escherichia coli yigP Gene Promoter

在已知大肠杆菌yigP基因的最小功能片段为yigP-P4P2基础上,将该片段装载于无外源启动子的载体质粒上,通过功能回补yigP基因缺陷株JDP14,发现其可以代替温敏质粒,保证大肠杆菌正常生长,推断其包含完整转录单元。RT-PCR结果显示该片段内部有转录产物,即yigP-P4P2片段具有转录独立性。将不同长度的yigP-P4P2亚片段克隆到启动子探针质粒pSP-Z上,通过对重组菌进行蓝白斑筛选及β-半乳糖苷酶活性测定,结果显示pP40V3-Z/JM83和pP40V4-Z/JM83可观测到蓝色菌斑,并检测到较弱的β-半乳糖苷酶活性,由此表明大肠杆菌yigP基因启动子位于该片段上游,下游边界位于引物V4区域内。

The yigP-P4P2 is the smallest known functional fragment of yigP gene in Escherichia coli.We cloned the yigP-P4P2 fragment into vector without exogenous promoter.After transferring the new plasmid into JDP14,a yigP gene defect Escherichia coli strain,the normal growth of Escherichia coli was observed.This observation showed that the plasmid played a similar role of the temperature-sensitive plasmid suggesting the plasmid contained intact transcriptional unit.We further verified the transcription products from sequence of this plasmid by RT-PCR,suggesting independent ability of transcription.We cloned a series of sub-yigP-P4P2 fragments with various lengths into promoter probe plasmid pSP-Z for blue white screening and β-galactosidase enzyme activity assay.The results showed that pP40V3-Z/JM83 and pP40V4-Z/JM83 were positive（blue） and β-galactosidase enzyme activity were detected among them as well.Based on these observations,we concluded the promoter of Escherichia coli yigP is located upstream of the yigP-P4P2 fragment;and the downstream boundary is located in sequence complementary to primer V4.