论文部分内容阅读
目的研究温度和酸度对田鼠型鼠疫耶尔森菌psa位点的转录调控作用。方法首先设计相关跨基因引物,用RT-PCR方法验证psa位点的相关操纵子结构。扩增操纵子首基因上游启动子区500 nt序列,并克隆入pRW50质粒β-半乳糖苷酶基因的上游,将重组质粒转入鼠疫菌中,通过测定β-半乳糖苷酶活性差异来判断不同温度(26和37℃)和酸度(pH 6.0和pH 8.0)对psaE和psaA的调控关系。最后提取鼠疫菌在上述不同温度和酸度条件下的总RNA,采用引物延伸实验进一步明确温度和酸度对psaA的调控关系。结果与结论 RT-PCR结果证实了田鼠型鼠疫菌的psa位点由操纵子psaEF和psaABC构成;半乳糖苷酶和引物延伸实验结果显示在37℃、pH 6.0培养条件下psaABC的转录水平最高,而psaEF的转录水平无明显变化,提示psaA的表达水平在37℃酸性条件下表达量最高,psaE则不受温度和酸度的调控。
Objective To study the transcriptional regulation of temperature and acidity on the psa locus of Microtus. Methods The cross-gene primers were designed and the related operon structure of psa site was verified by RT-PCR. A 500 nt sequence was amplified from the upstream of the promoter region of the operon and cloned into the upstream of the β-galactosidase gene of pRW50 plasmid. The recombinant plasmid was then transferred into Yersinia pestis to determine the difference in β-galactosidase activity The regulation of psaE and psaA at different temperatures (26 and 37 ℃) and acidity (pH 6.0 and pH 8.0). Finally, the total RNA of Y. pestis at different temperatures and acidities was extracted, and the primer-extension experiment was used to further clarify the regulation of temperature and acidity on psaA. RESULTS AND CONCLUSION: The results of RT-PCR confirmed that the psa site of V. simonii was composed of the operon psaEF and psaABC. The results of galactosidase and primer extension showed that the transcription level of psaABC was highest at 37 ℃ and pH 6.0, However, there was no significant change in the transcriptional level of psaEF, suggesting that the expression level of psaA was highest under acidic conditions at 37 ℃ and psaE was not regulated by temperature and acidity.