沙门氏菌sRNA RybB及伴侣蛋白Hfq对孔蛋白OmpW表达的调控作用  

Regulation of sRNA RybB and Chaperone Hfq of Salmonella on Expression of Porin OmpW

作　　者：令狐远凤 潘永 杨阳 段世宇 张家莉 杨琦[1,2] LINGHU Yuanfeng;PAN Yong;YANG Yang;DUAN Shiyu;ZHANG Jiali;YANG Qi(College of Animal Science,Guizhou University,Guiyang,Guizhou 550025;Institute of Animal Diseases,Guizhou University,Guiyang,Guizhou 550025;Institute of Animal Husbandry and Veterinary Medicine,Guizhou Academy of Agricultural Sciences,Guiyang,Guizhou 550025)

机构地区：[1]贵州大学动物科学学院,贵州贵阳550025 [2]贵州大学动物疫病研究所,贵州贵阳550025 [3]贵州省农业科学院畜牧兽医研究所,贵州贵阳550025

出　　处：《中国家禽》2023年第6期30-35,共6页China Poultry

基　　金：国家自然科学基金项目(32260884)。

摘　　要：为探究sRNA RybB和伴侣蛋白Hfq对沙门氏菌孔蛋白OmpW表达的调控作用,研究利用λred同源重组系统构建ompW基因lacZ融合菌株,再通过P22噬菌体转导技术构建颜色指示菌株中rybB和hfq相关基因的单缺失株、双缺失株,通过β-半乳糖苷酶活性试验和qPCR分析,确定sRNA RybB及伴侣蛋白Hfq对孔蛋白OmpW表达的调控作用。结果显示:成功构建了颜色指示菌株ΔompW∶∶LacZ和相对应的基因缺失菌株;与标准株LT2相比,ΔhfqY25A、ΔhfqK56A、Δhfq65、Δhfq87基因使ompW蛋白表达水平极显著下调(P<0.01),具有正调控作用,而Δhfq72、Δhfq∶∶tet、ΔrybB、ΔrybBΔhfq∶∶tet基因使ompW蛋白表达水平极显著上调(P<0.01),具有负调控作用;与标准株LT2相比,缺失hfq定点突变和rybB对ompW基因具有负调控作用。研究表明,沙门氏菌伴侣蛋白Hfq及RybB对孔蛋白ompW基因的蛋白表达水平和转录水平产生不同程度的影响,初步阐明了伴侣蛋白Hfq、sRNA RybB和靶基因三者的内在关系,为沙门氏菌减毒活疫苗与新型抗菌药物研制的研究奠定基础。To investigate the regulatory effect of sRNA RybB and chaperone Hfq on the expression of Salmonella porin OmpW,λRed homologous recombination system was used to construct ompW and lacZ fusion strains in this study,single dele⁃tion and double deletion of rybB and hfq related genes in color indicator strains were constructed by P22 phage transduction technology,β-galactosidase activity test and qPCR were used to determine the regulatory effect of sRNA RybB and chaperone Hfq on the expression of porin OmpW.The results showed that the color indicator strain and the corresponding gene deletion strain was successfully constructed.Compared with standard strain LT2,ΔhfqY25A,ΔhfqK56A,Δhfq65,Δhfq87 genes significantly de⁃creased ompW protein expression level(P<0.01),which showed a positive regulatory effect.WhileΔhfq72,Δhfq∶∶tet,ΔrybB,ΔrybBΔhfq∶∶tet genes significantly increased ompW protein expression level(P<0.01),which showed a negative regulatory effect.The qPCR results showed that deletion of site-directed mutation of hfq and rybB had negative regulation effect on ompW gene compared with standard strain LT2.The results showed that the chaperone proteins Hfq and RybB of Salmonella had different ef⁃fects on protein expression level and transcription level of porin ompW gene,and preliminarily clarified the internal relationship between chaperone proteins Hfq,sRNA RybB and target genes,and laid a foundation for the research of attenuated live vaccine and new antibacterial drugs against Salmonella.

关 键 词：沙门氏菌 λ-Red同源重组系统 sRNA RybB 伴侣蛋白Hfq OmpW 

分 类 号：S855.1[农业科学—临床兽医学]