鼠伤寒沙门氏菌STM LT2 Hfq关键作用位点的分析

Analysis of Key Action Sites of Salmonella Typhimurium STM LT2 Hfq

为探究伴侣蛋白Hfq与GcvB及其靶基因oppA可能存在的关键作用位点,本研究通过λ-Red同源重组技术构建Hfq远端面和近端面核心氨基酸的突变菌株及Hfq C-端截短菌株,在此基础上构建相应的Hfq-His6标签蛋白标签融合菌株。运用P22噬菌体转导技术构建相应的GcvB基因缺失菌株以及oppA::lacZ基因融合菌株。通过Western blotting试验检测Hfq蛋白水平,实时荧光定量PCR检测GcvB和oppA基因转录水平,β-半乳糖苷酶试验检测oppA蛋白水平。Western blotting结果显示,Hfq远端面和近端面核心氨基酸的突变及Hfq C-端的截短均不同程度地上调了Hfq蛋白水平。实时荧光定量PCR结果显示,与对照组相比,Hfq近端面核心氨基酸的突变使GcvB基因转录水平下调。β-半乳糖苷酶试验结果显示,与对照组相比,Hfq远端面和近端面核心氨基酸的突变及Hfq C-端的截短均未造成oppA基因转录水平的明显变化,但均不同程度地上调了oppA蛋白水平,其中Hfq近端面核心氨基酸突变及Hfq C-端分别截短至第65和72位氨基酸时oppA蛋白水平上调最为明显,分别上调了2.9、3.3和2.0倍。以上结果表明,Hfq近端面对于Hfq维持GcvB稳定性非常重要,Hfq近端面第56位氨基酸及Hfq C-端第65-87位氨基酸与oppA蛋白水平呈负相关。

To explore the possible key action sites of Hfq,GcvB and their target gene oppA,in this study,λ-Red homologous recombination technology was used to construct mutant strains with core amino acids on the distal and proximal faces of Hfq and Hfq C-terminal truncated strains.On this basis,the corresponding Hfq-His6 protein tag fusion strains were constructed.P22 phage transduction technology was used to construct corresponding GcvB gene deletion strain and oppA::lacZ gene fusion strains.The protein level of Hfq was detected by Western blotting test,the transcription level of GcvB and oppA gene was detected by Real-time quantitative PCR,and the level of oppA protein was detected byβ-galactosidase test.Western blotting test results showed that the mutation of core amino acids on the distal and proximal faces of Hfq and the truncation of the C-terminus of Hfq all increased the protein level of Hfq to varying degrees.The results of Real-time quantitative PCR showed that compared with control group,the mutation of the core amino acid of the proximal face of Hfq down-regulated the transcription level of the GcvB gene.The results ofβ-galactosidase test showed that compared with control group,the mutations of core amino acids on the distal and proximal faces of Hfq and the truncation of the C-terminus of Hfq did not cause significant changes in the transcription level of oppA gene,but they all increased the level of oppA protein to varying degrees.Among them,when the core amino acid mutation of Hfq proximal face and the C-terminus of Hfq were truncated to amino acid at positions 65 and 72,the protein level of oppA was up-regulated most obviously,up-regulating 2.9,3.3 and 2.0 times,respectively.The results indicated that the proximal face of Hfq was very important for Hfq to maintain the stability of GcvB,the amino acid at position 56 of the proximal face of Hfq and the amino acid at positions 65 to 87 of the C-terminus of Hfq were negatively correlated with the level of oppA protein.