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目的从转录水平上研究鼠疫耶尔森菌Fur蛋白对waaA的转录调控机制。方法提取鼠疫菌野生株(WT)和fur突变株(Δfur)的总RNA,采用引物延伸实验研究waaA的转录起始位点,并根据产物的丰度判断Fur蛋白对waaA的调控关系。构建克隆有waaA上游启动子区的LacZ重组质粒,将重组质粒转入WT和Δfur中,通过测定β-半乳糖苷酶活性来比较两株重组菌中的waaA启动子活性,进一步验证Fur蛋白对waaA的调控关系。PCR扩增waaA启动子区DNA序列全长,并纯化鼠疫菌His-Fur蛋白,通过体外的凝胶阻滞实验,即电泳迁移率变动实验(EMSA)和DNaseⅠ足迹实验验证His-Fur蛋白对waaA启动子区是否具有直接的结合作用。结果与结论 DNaseⅠ足迹实验表明,Fur蛋白对waaA启动子区的结合位置为-204至-150之间的碱基(翻译起始位点为+1);引物延伸实验显示waaA有一个转录起始位点T(-26),且该位点的转录活性直接被Fur蛋白激活。
AIM To investigate the transcriptional regulation of waaA by Fur protein of Yersinia pestis at the transcriptional level. Methods The total RNA of Y. pestis WT strain and fur mutant strain (Δfur) was extracted. The transcriptional start site of waaA was studied by primer extension and the regulation of Fur protein to waaA was determined according to the abundance of the product. Construction of lacZ recombinant plasmid cloned waaA upstream promoter region, the recombinant plasmid was transferred to WT and Δfur, β-galactosidase activity was measured to compare the two recombinant strains of waaA promoter activity to further verify the Fur protein pair waaA regulatory relationship. PCR was used to amplify the full-length DNA sequence of waaA promoter region and purify the His-Fur protein of Yersinia pestis. His-Fur protein was verified by EMSA and DNase I footprinting experiments in vitro. Whether the promoter region has a direct binding. RESULTS AND CONCLUSION DNase I footprinting showed that the binding site of Fur protein to the waaA promoter region was between -204 and -150 (the translation initiation site was +1); primer extension experiments showed that waaA had a transcription initiation Site T (-26), and the transcriptional activity of this site is directly activated by Fur protein.