鼠伤寒沙门菌小RNA RybB及伴侣蛋白Hfq对孔蛋白OmpD的表达调控

Small RNA RybB and chaperone protein Hfq of Salmonella Typhimurium regulate expression of porin OmpD

【目的】探究小RNA(small RNA,sRNA)RybB和伴侣蛋白Hfq对沙门氏菌孔蛋白OmpD表达的调控作用。【方法】以鼠伤寒沙门菌(Salmonella Typhimurium,STM)为研究对象,将含有编码β-半乳糖苷酶的lacZ报告基因的pCE40质粒转入ompD基因单缺失菌株中以获得lacZ报告菌株;在此基础上,利用P22噬菌体转导技术分在lacZ报告菌株中构建rybB全序列缺失、hfq全序列缺失、hfq点序列敲除和hfq序列截短以获得双突变实验菌株,以及rybB全序列缺失和hfq全序列缺失的三突变实验菌株。通过β-半乳糖苷酶活性试验和RT-qPCR探究sRNA RybB及伴侣蛋白Hfq对孔蛋白OmpD表达的调控作用。【结果】成功在lacZ报告菌中构建rybB全序列缺失、hfq全序列缺失、hfq点序列敲除和hfq序列截短等双缺失实验菌株,以及rybB全序列缺失和hfq全序列缺失的三突变实验菌株。与野生型(wild type,WT)菌株相比,在lacZ报告菌株中,hfq基因截短为87个氨基酸序列的突变株中的OmpD蛋白活性下调了2.16%,其余实验株OmpD蛋白的β-半乳糖苷酶活性均呈上升趋势。与WT菌株相比,实验菌株ompD基因转录水平除了STM LT2ΔompD::lacZΔhfq65的上调不具有显著性外,其余实验菌株均显著(P<0.05)上调,其中lacZ报告菌株、rybB全序列缺失和hfq全序列缺失的三突变实验菌株ompD基因转录水平上调最明显,为1.83倍。【结论】ompD基因的转录及蛋白表达主要受hfq基因和sRNA RybB的负反馈调节;Hfq的远端面在对ompD基因的转录抑制中起关键作用。通过多个基因突变菌株的构建,阐述了sRNA伴侣蛋白Hfq与孔蛋白OmpD的相互作用关系,探索了Hfq对ompD的调控关键区域,丰富了sRNA的调控理论。

[Objective]To investigate the role of the small RNA(sRNA)RybB and the chaperone protein Hfq in regulating the expression of porin OmpD in Salmonella.[Methods]In this study,Salmonella Typhimurium(STM)was used as the research object.The pCE40 plasmid carrying the reporter gene lacZ encodingβ-galactosidase was transferred into the single mutant lacking ompD to obtain the lacZ reporter strain.On this basis,we employed P22 phage-mediated transduction to construct the double mutants lacking full-length rybB,full-length hfq,partial sequence of hfq,or truncated hfq sequence and the triple mutantlacking full-length rybB and full-length hfq.The regulatory effects of RybB and Hfq on the expression of OmpD were probed byβ-galactosidase activity assay and RT-qPCR.[Results]We successfully constructed the double and the triple mutant.Compared with that in the wild type(WT),the OmpD activity was down-regulated by 2.16%in the lacZ reporter strain with truncated sequence(87 residues)of hfq,and theβ-galactosidase activity of OmpD increased in the rest strains.Compared with WT,except for STM LT2ΔompD::lacZΔhfq6,all the mutants showed up-regulated transcript level of ompD(P<0.05),with the most significant up-regulation of 1.83-folds in the triple mutant.[Conclusion]The transcription and translation of ompD are mainly regulated by the negative feedback of hfq and RybB.The distal end of Hfq plays a key role in the transcriptional repression of ompD.By construction of several mutants,this article illustrated the interactions of RybB and Hfq with OmpD and explored the key regions of Hfq in regulating ompD,which enriched the theory of sRNA regulation.