作　　者： (王净）; (韩延平）; (杨瑞馥）;

机构地区： 河北北方学院动物科技学院,张家口075131 军事医学科学院微生物流行病研究所病原微生物生物安全国家重点实验室,北京100071

出　　处： 《科学通报》 2017年第24期2804-2813,共10页

摘　　要： 为了确定单分子Sgr S的空间定位,在野生株Escherichia coli MG1655基础上,构建敲除株(35)sgr S和过表达株(35)sgr S-p BAD-Sgr S,并以3株菌为供试菌株建立了定位大肠杆菌Sgr S的单分子荧光原位杂交(sm FISH)方法.优化条件为:杂交后细菌洗涤次数为5次,涂片时加入Slow Fade Diamond Antifade Mountant防淬灭,细菌在杂交液和探针的混合液中经40℃杂交3 h,杂交前探针置98℃变性10 min,Alexa-555 WGA染料用来标记细胞壁.sm FISH操作步骤确定为:固定-洗涤-透化-预杂交-杂交-洗涤-染细胞壁-洗涤-涂片,最后使用N-SIM超分辨率显微镜观察成像.定位结果显示,Sgr S呈绿色点状荧光弥散分布于细菌胞浆中,Alexa-555 WGA染料标记细胞壁呈红色,从而完整地定位了大肠杆菌的形态.sm FISH方法和超分辨显微技术的结合可为深入研究细菌模式基因s RNA的定位以及s RNA介导的调控机制提供线索和技术支持. Single-cell gene expression studies could not only provide the real-time information on intracellular amount and subcellular localization of RNA, but also could reveal the regulatory mechanism of bacterial variation, which cannot be obtained based on traditional view of bacterial populations. Owing to the application of green fluorescent protein （GFP） reporters, most of studies have mainly focused on proteins. Recently, the quantitative and localization of bacterial RNAs have been also implemented at single transcript level. All of the non-coding RNAs who function as crucial regulators of gene expression are called regulatory small RNAs （sRNA） in bacteria. Single molecular imaging techniques for labeling bacterial sRNA can be used to detect the activity and abundance of sRNA, which will help us to understand its regulatory mechanism and biological function. SgrS, a conservative sRNA in ＂/-proteobacteria, is highly expressed under glucose-phosphate stress and involve in the sugar metabolism in bacteria. We chose SgrS as a model to establish the single-molecule fluorescence in situ hybridization （smFISH） method of RNA detection at the single-molecule level in Escherichia coli. It will be helpful for further studies on sRNA subcellular localization and gene regulation. We used kanamycin resistance box replace sgrS to successful construct sgrS deletion mutant AsgrS and subsequently cured the helper plasmid pKD46. The SgrS overexpression strain AsgrS-pBAD-SgrS was generated by introducing SgrS-overexpressed plasmid into the AsgrS strains. E. coli MG1655, AsgrS and AsgrS-pBAD-SgrS, all E. coli K-12 derivatives, were used in this study. The smFISH method on SgrS in E. coli was established. The optimal conditions are briefly stated as follows. The cells were fixed in 3.7% formaldehyde at room temperature for 30 minutes. Ceils were permeabilized at 4~C for overnight. Washed cells with 40% wash solution was resuspended in 40% hybridization buffer. The cells were centrifuged and resuspended in the hybridizat

关 键 词： 大肠杆菌 单分子荧光原位杂交 超分辨率显微镜