摘要
细菌调节小RNA通常与靶mRNA结合,影响翻译和mRNA降解过程.了解细菌小RNA的定量和定位信息,将有助于揭示细菌转录后水平的调控机制.小RNA SgrS通过抑制ptsG mRNA翻译起始,参与细菌磷酸葡萄糖代谢的应激过程.本研究应用单分子荧光原位杂交(smFISH)方法和超分辨显微技术可视化定位大肠杆菌细胞内小RNA SgrS,并初步验证伴侣分子Hfq蛋白和RNaseE降解酶对小RNA SgrS定位的影响.选取大肠杆菌模式菌MG1655 (野生株)、sgrS敲除株(△sgrS)和过表达株(△sgrS-pBAD-SgrS),使用RNA印迹和smFISH方法验证SgrS的过表达.应用smFISH方法分别在野生菌株、hfq敲除株(△hfq)和rne敲除株(△rne-710)中定位小RNA SgrS和ptsG mRNA,超分辨成像观察.与野生株相比,△hfq和△rne-710中SgrS主要定位于近膜区域,ptsG mRNA定位于细菌胞浆中,并且这两种RNA拷贝数均极显著增加.以上结果表明,分别敲除大肠杆菌hfq和rne-710基因导致SgrS和ptsG mRNA的表达量增加. smFISH方法和超分辨技术的结合应用为细菌RNA的直观定量和定位提供了高敏感性的检测方法,可用于基因调控的功能研究.
Bacterial small regulatory RNAs could influence the translation and/or mRNA degradation by binding with target mRNA.It is helpful to reveal the regulatory mechanism of bacterial post-transcriptional level for knowing quantitative and localization information of bacterial small RNA(sRNA).Small RNA SgrS is participated in the stress process of bacterial glucose phosphate metabolism by inhibiting ptsG mRNA translation initiation.In this study,Escherichia coli intracellular sRNA SgrS was located visually by smFISH method and super-resolution microscopy technology,and the effects of chaperone Hfq protein and RNase E degrading enzyme on the localization of sRNA SgrS were verified preliminarily.Over-expression of SgrS in E.coli model strain MG1655(wild strain),sgrS knockout strain(△sgrS)and over-expression strain(△sgrS-pBAD-SgrS)were validated by Northern blot and smFISH methods.sRNA SgrS and ptsG mRNA were located respectively in the wild-type strain,the hfq knockout strain(△hfq)and the rne knockout strain(△rne-710)by smFISH method.Comparing with the wild-type strain,SgrS was mainly located nearby cell membrane,ptsG mRNA was located in bacterial cytoplasm in△hfq and△rne-710 strains by super-resolution imaging,and the copy numbers of both RNA were significantly increased(P<0.01).These results suggest that the expression levels of SgrS and ptsG mRNA were increased significantly in hfq knockout E.coli and rne-710 knockout E.coli.Integrated application of smFISH method and super-resolution technology found a highly sensitive detection method for the intuitive quantification and localization of bacterial RNA,which can be used for the research of gene regulation functional.
作者
王净
阮崇美
白园园
韩延平
杨瑞馥
WANG Jing;RUAN Chong-Mei;BAI Yuan-Yuan;HAN Yan-Ping;YANG Rui-Fu(College of Animal Science and Technology,Hebei North University,Zhangjiakou 075131,China;Beijing Institute of Microbiology and Epidemiology,Beijing 100071,China;School of Animal Science and Veterinary Medicine,Xinyang Agriculture and Forestry University,Xinyang 464000,China)
出处
《生物化学与生物物理进展》
SCIE
CAS
CSCD
北大核心
2019年第4期415-422,共8页
Progress In Biochemistry and Biophysics
基金
国家重点基础研究发展计划(973计划)(2014CB744405)
河北省重点研发计划(18236609D)资助项目~~
