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
发布时间：2018-09-06 13:55
【摘要】：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。
[Abstract]:In order to study the effect of (UTR) on the expression of flhDC, the genomic DNA of Salmonella typhimurium 542 (STM542) was used as a template. The full-length flhDC genes with different 5'-UTR lengths (5 species) were amplified and cloned into plasmid PBAD33 containing arabinose promoter. The effect of different 5'-UTR regulatory sequences on the expression of flhDC gene was evaluated by measuring the diameter of Escherichia coli colony containing different length of flhDC gene on the semisolid plate containing arabinose. In order to accurately determine the activity differences of different regulatory sequences, we further constructed a T vector using lacZ as a reporter gene, and cloned the amplified fragments corresponding to the first four flhDC gene regulatory sequences into the constructed T vector. The activity parameters of 尾-galactosidase were obtained by measuring the 尾-galactosidase activity of 尾-galactosidase. The results showed that under arabinose induction, the flhDC gene fragment corresponding to 1572bp had the lowest regulatory sequence activity, and the flhDC gene fragment corresponding to 1201bp had the highest regulatory sequence activity. This study laid a foundation for further study on the regulatory function of flhDC gene.
【作者单位】： 浙江大学动物科学技术学院;
【基金】：国家自然科学基金(31272582、30571380)
【分类号】：S852.61
[Abstract]:In order to study the effect of (UTR) on the expression of flhDC, the genomic DNA of Salmonella typhimurium 542 (STM542) was used as a template. The full-length flhDC genes with different 5'-UTR lengths (5 species) were amplified and cloned into plasmid PBAD33 containing arabinose promoter. The effect of different 5'-UTR regulatory sequences on the expression of flhDC gene was evaluated by measuring the diameter of Escherichia coli colony containing different length of flhDC gene on the semisolid plate containing arabinose. In order to accurately determine the activity differences of different regulatory sequences, we further constructed a T vector using lacZ as a reporter gene, and cloned the amplified fragments corresponding to the first four flhDC gene regulatory sequences into the constructed T vector. The activity parameters of 尾-galactosidase were obtained by measuring the 尾-galactosidase activity of 尾-galactosidase. The results showed that under arabinose induction, the flhDC gene fragment corresponding to 1572bp had the lowest regulatory sequence activity, and the flhDC gene fragment corresponding to 1201bp had the highest regulatory sequence activity. This study laid a foundation for further study on the regulatory function of flhDC gene.
【作者单位】： 浙江大学动物科学技术学院;
【基金】：国家自然科学基金(31272582、30571380)
【分类号】：S852.61
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