鼠伤寒沙门氏菌LT2 Hfq与fadL、ompN、ybfM mRNA 5′UTR结合位点的预测与验证分析  

Analysis of Hfq-binding site in the fadL, ompN and ybfM mRNA 5′UTR of Salmonella typhimurium LT2

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

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：《福建农林大学学报（自然科学版）》2022年第5期636-643,共8页Journal of Fujian Agriculture and Forestry University:Natural Science Edition

基　　金：国家自然科学基金项目(31760740);贵州省研究生教育创新计划项目(GZZ2017002).

摘　　要：通过5′RACE技术分析fadL、ompN、ybfM mRNA的5′UTR,并筛查fadL、ompN、ybfM mRNA 5′UTR中Hfq结合偏好型基序(ARN)n.利用λ-Red同源重组酶和FLP重组酶系统构建fadL、ompN、ybfM基因(ARN)n基序缺失基础上的lacZ基因融合菌株,运用P22噬菌体转导技术构建相应的hfq基因缺失菌株,通过β-半乳糖苷酶试验检测重组菌株的蛋白水平.结果显示,fadL中筛选出ARN-1(AAAAAA)、ARN-2(AAAAATAAT),其缺失降低了fadL的蛋白表达量,分别降低72%、56%;ompN中筛选出ARN-1(GTTTTT)、ARN-2(TCTTTT)、ARN-4(TTTGCT),其缺失提高了ompN的蛋白表达量,分别提高了1.29、1.30、1.07倍,ARN-3(ATTATT)缺失使ompN的蛋白表达量降低了10%;ybfM中筛选出ARN-1(AAGAGG)、ARN-2(AGCAAT)、ARN-3(AGTAAA)、ARN-4(AAAAATAGT)、ARN-5(AACAGAAAG),其缺失均增加了ybfM蛋白水平变化,分别为4.20、3.99、10.90、227.00、46.00倍.结果表明,ybfM、ompN、fadL中(ARN)n位点不同缺失能导致ybfM、ompN、fadL表达水平出现上调或下调,部分(ARN)n序列缺失可影响Hfq对相应靶基因蛋白水平调控作用.The 5′untranslated region(5′UTR) of fadL, ompN and ybfM mRNAs were analyzed by 5′RACE technology aiming to screen the(ARN)n of chaperonin Hfq. The lacZ fusion strains with the deletion of(ARN)n in the fadL, ompN or ybfM gene were constructed by λ-red homologous recombinase and FLP recombinase systems, which was followed by the deletion of hfq gene by P22 phage transduction. Protein expression differences in the recombinant strains were detected by β-galactosidase assay. The results showed that the deletion of ARN-1(AAAAAA)or ARN-2(AAAAATAAT) in fadL reduced the expression of fadL by 72% and 56%, respectively. The deletion of ARN-1(GTTTTT), ARN-2(TCTTTT) or ARN-4(TTTGCT) in ompN increased the expression of ompN by 1.29, 1.30 and 1.07 times respectively, while the deletion of ARN-3(ATTATT) led to the decrement by 10%. The deletion of ARN-1(AAGAGG), ARN-2(AGCAAT), ARN-3(AGTAAA), ARN-4(AAAAATAGT) or ARN-5(AACAGAAAG) in ybfM resulted in 4.20, 3.99, 10.90, 227.00 and 46.00 times higher expressions, respectively. To summarize, the deletions of various(ARN)n sites in ybfM, ompN and fadL lead to up or down-regulation of their expressions, and the deletion of some(ARN)n may affect the regulation of Hfq on the expression of targeting genes.

关 键 词：沙门氏菌 λ-red同源重组系统 伴侣蛋白Hfq 外膜蛋白 

分 类 号：S855.1[农业科学—临床兽医学]