鼠伤寒沙门菌伴侣蛋白Hfq与小RNA GcvB结合位点的初步分析  

Preliminary Exploration of Binding Sites of Salmonella Typhimurium Chaperone Protein Hfq on Small RNA GcvB

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

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

出　　处：《畜牧兽医学报》2022年第11期4110-4115,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

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

摘　　要：旨在探究Hfq与GcvB可能的作用结合位点,本研究首先筛查GcvB中Hfq结合偏好型富U基序。通过λ-Red同源重组酶系统构建GcvB富U基序突变或截短菌株,构建GcvB终止子茎延伸菌株。运用P22噬菌体转导技术构建相应的hfq基因缺失菌株以及oppA::lacZ基因融合菌株。通过qRT-PCR检测重组菌株的gcvB基因转录水平,β-半乳糖苷酶试验检测oppA基因编码蛋白水平。预测结果显示,GcvB存在2个Hfq结合偏好型富U基序U_(5)和U_(8)。qRT-PCR结果显示,U_(5)基序的突变未造成gcvB基因转录水平明显的变化,而gcvB基因转录水平随U_(8)基序的截短而下调,截短为U_(5)和U_(4)时下调最为明显,分别下调40.5%和37.5%。延伸U_(4)截短菌株的GcvB终止子茎,结果发现,gcvB基因转录水平几乎恢复到了对照组的水平,同时也发现,尽管U_(4)截短菌株gcvB基因转录水平得以恢复,却丧失了Hfq协同GcvB转录后负调控oppA mRNA的能力。以上结果表明,U_(5)基序与Hfq维持GcvB稳定性无明显关联。U_(8)基序对Hfq协助GcvB转录后负调控oppA mRNA以及维持GcvB稳定性是重要的,推测其为Hfq与GcvB的作用结合位点。To explore the possible binding sites of Hfq on GcvB,in this study,the Hfq binding preference U-rich motif in GcvB was screened.The mutant and truncated strains of the GcvB U-rich motif were constructed by the λ-Red homologous recombinase system,and the GcvB terminator stem extension strain was constructed at the same time.P22 phage transduction technology was used to construct corresponding hfq gene deletion strains and oppA::lacZ gene fusion strains.The gcvB gene transcription expression level of the recombinant strain was detected by qRT-PCR,and the oppA gene protein expression level was detected by the β-galactosidase test.The results showed that the mutation of U_(5) motif did not cause obvious changes in the transcription and expression level of gcvB gene,but when the U_(8) motif was truncated to U_(5) and U_(4),it was reduced by 40.5% and 37.5%,respectively.After the GcvB terminator stem of the U_(4) truncated strain was extended,it was discovered that the transcription and expression level of gcvB gene was restored,but the ability to Hfq negatively regulating oppA mRNA was weakened after co-transcription.The above results show that the U_(5) motif has no obvious relationship with Hfq maintaining the stability of GcvB,but the U_(8) motif is important for Hfq to assist GcvB with regulating oppA mRNA negatively after co-transcription and maintain GcvB stability.In summary,we speculate that the U_(8) motif is the binding site of Hfq on GcvB.

关 键 词：沙门菌 λ-Red同源重组系统 GcvB 伴侣蛋白Hfq oppA 

分 类 号：S855.1[农业科学—临床兽医学]