一种lacZ报告基因T载体的构建及其在沙门氏菌鞭毛主调控基因flhDC表达活性测定中的应用

Construction of T-vector with lacZ reporter gene and its application in the expression activity of flagellar master regulatory gene flhDC in Salmonella

为研究5'-非翻译区(UTR)对沙门氏菌鞭毛主调控基因flhDC表达的影响,本研究以鼠伤寒沙门氏菌542(STM542)基因组DNA为模板,扩增含不同长度5'-UTR的flhDC全长基因(共5种),并将其克隆至含阿拉伯糖启动子的质粒PBAD33中。通过测定含阿拉伯糖的半固体平板上包含不同长度flhDC基因的重组大肠杆菌菌落的直径,初步评估不同5'-UTR调控序列对flhDC基因表达的影响。为精确测定不同调控序列的活性差异,本实验室进一步构建了以lacZ为报告基因的T载体,并将扩增的对应于前4种flhDC基因调控序列片段克隆于构建的T载体,通过测定其β-半乳糖苷酶活性获得相应调控序列的活性参数。结果显示,在阿拉伯糖诱导下,对应于1572bp的flhDC基因片段的调控序列活性最低,对应于1201bp的flhDC基因片段的调控序列活性最高,本实验为进一步研究flhDC基因的调控功能奠定基础。

In order to investigate the effect of the 5＇- untranslated region （UTR） on the expression activity of flagellar master regulatory gene tlhDC in Salmonella. The 5 flhDC genes with different lengths of 5＇-UTR were amplified from the genome DNA in Salmonella typhimurium 542, and were cloncd into plasmid of pBAD33 with Arabia suqar promoter. The motility assay was operated in E.coli which was used to evaluated the effcct of the different lengths of 5 ＇-UTR on the expression of flhDC genes. The 4 regulatory sequences corresponding to the first four different lengths tlhDC genes were connected to a T-vector with lacZ report gene constructed by our laboratory and the β-galactosidase activity test were experimented in E.coli to further research their activity difference. The results of the above two experiments showed that the activity of regulatory sequence corresponding to the 1,572 bp flhDC was lowest and the regulatory sequence corresponding to the 1,201 bp f/hDC was highest. This study established basis for fitrther research on the regulation function of the t/hDC gene in Salmonella.