单细胞显微注射技术在原生动物草履虫研究中的应用  

Application of the Microinjection Technology in the Research of Paramecium cells

作　　者：史磊 申香玉 申远 SHI Lei;SHEN Xiang-Yu;SHEN Yuan(Department of Biochemistry and Molecular Biology,School of Basic Medical Science,Xinxiang Medical University,Xinxiang 453003,Henan,China;Department of Traditional Chinese Medicine,School of Pharmacy,Xinxiang Medical University,Xinxiang 453003,Henan,China)

机构地区：[1]新乡医学院基础医学院生物化学与分子生物学教研室,河南新乡453003 [2]新乡医学院药学院中药学教研室,河南新乡453003

出　　处：《中国生物化学与分子生物学报》2020年第12期1508-1513,共6页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金项目(No.31500982)资助。

摘　　要：对大多数单细胞原生动物的分子遗传学研究,目前还缺乏行之有效的转基因手段。针对这种情况,本研究以第四双小核草履虫为对象,将包含自身调控序列的草履虫纤毛内运输蛋白(intraflagellar transport,IFT)基因IFT43或IFT46插入表达载体Pzz02-GFP中,用单细胞显微注射术将重组质粒注入到第四双小核草履虫营养大核内。蛋白质免疫印记结果显示,与绿色荧光蛋白(green fluorescent protein,GFP)标签偶联的IFT43或IFT46在草履虫细胞中表达,利用荧光显微镜或激光共聚焦成像系统观察,IFT43-GFP与IFT46-GFP融合蛋白显示出相似的纤毛定位,同时它们在草履虫多纤毛内的运输过程被动态记录。本研究表明,显微注射方法能够在草履虫细胞中实现外源基因转入和表达,在对单细胞模式生物的研究中具有广泛的应用前景。We developed a microinject method in the unicellular Paramecium model to establish a proper transgene methodology for protists for genetic research,.In this study,intraflagellar transport(IFT)genes including IFT46 and IFT43 with its own promoter were cloned into Pzz02-GFP plasmids,then recombinant plasmids were transformed into nutrient macronuclear of Paramecium tetraurelia by microinjection.The result of Western blotting shows that IFT46-GFP or IFT43-GFP fusion proteins were produced in Paramecium cells.By fluorescence or confocal microscopy,we record the bi-directional movements of IFT proteins along cilia,which confirms the efficiency of the microinjection technology in transforming exogenous genes to unicellular models like Paramecium.

关 键 词：显微注射技术 草履虫 单细胞模型 外源基因表达 

分 类 号：Q3[生物学—遗传学]