一種lacZ報告基因T載體的構(gòu)建及其在沙門氏菌鞭毛主調(diào)控基因flhDC表達活性測定中的應用
發(fā)布時間：2018-09-06 13:55
【摘要】：為研究5'-非翻譯區(qū)(UTR)對沙門氏菌鞭毛主調(diào)控基因flhDC表達的影響,本研究以鼠傷寒沙門氏菌542(STM542)基因組DNA為模板,擴增含不同長度5'-UTR的flhDC全長基因(共5種),并將其克隆至含阿拉伯糖啟動子的質(zhì)粒PBAD33中。通過測定含阿拉伯糖的半固體平板上包含不同長度flhDC基因的重組大腸桿菌菌落的直徑,初步評估不同5'-UTR調(diào)控序列對flhDC基因表達的影響。為精確測定不同調(diào)控序列的活性差異,本實驗室進一步構(gòu)建了以lacZ為報告基因的T載體,并將擴增的對應于前4種flhDC基因調(diào)控序列片段克隆于構(gòu)建的T載體,通過測定其β-半乳糖苷酶活性獲得相應調(diào)控序列的活性參數(shù)。結(jié)果顯示,在阿拉伯糖誘導下,對應于1572bp的flhDC基因片段的調(diào)控序列活性最低,對應于1201bp的flhDC基因片段的調(diào)控序列活性最高,本實驗為進一步研究flhDC基因的調(diào)控功能奠定基礎。
[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.
【作者單位】： 浙江大學動物科學技術(shù)學院;
【基金】：國家自然科學基金(31272582、30571380)
【分類號】：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.
【作者單位】： 浙江大學動物科學技術(shù)學院;
【基金】：國家自然科學基金(31272582、30571380)
【分類號】：S852.61
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