粟酒裂殖酵母中指示线粒体形态的荧光标签的构建

Construction of Fluorescent Tag Indicating Mitochondrial Morphology in Schizosaccharomyces pombe

粟酒裂殖酵母是优秀的研究线粒体模型机理的模式生物。为建立适合粟酒裂殖酵母的指示线粒体形态的荧光标签,本研究以pYJ19质粒为骨架,构建了由粟酒裂殖酵母nmt41启动子控制的、以增强型绿色荧光EGFP为报告基因、以粟酒裂殖酵母自身蛋白Cox4为线粒体定位序列(Mitochondrial targeting sequences)来源的mts和以leu1营养为选择标记的质粒系统pMTS7。将pMTS7转化粟酒裂殖酵母yHL6381,以Mito-Tracker线粒体染料为对照,荧光显微镜观察结果发现,pMTS7能够与MitoTracker共定位,能正确显示线粒体定位。与具有一定毒性且染色结果不易控制的MitoTracker相比,利用pMTS7标记线粒体可以在显微镜下长时间观察清晰的线粒体形态,而且可以看到线粒体分裂融合的动态变化。pMTS7的成功构建为研究粟酒裂殖酵母中线粒体相关基因的突变体提供了技术工具。

Schizosaccharomyces pombe is an excellent model organism for studying mitochondrial mechanism.To establish a suitable fluorescent tag indicating the mitochondrial morphology for S.pombe,the plasmid system pMTS7 which was boned on plasmid pYJ19 with a nmt41 promoter,EGFP as reporter,the mts derived from S.pombe's own protein Cox4 as a mitochondrial targeting sequences and leu1 as selection marker,respectively,was constructed.pMTS7 was transformed into S.pombe named yHL6381,andMitoTracker was used as a control.The results of fluorescence microscopy showed that pMTS7 could be co-located withMitoTracker and correctly display mitochondrial localization.Compared with the MitoTracker which has some toxic and is difficult to control,using pMTS7 to mark mitochondria can observe clear mitochondrial morphology under a microscope for a long time,and can see the dynamic changes of mitochondrial fission and fusion.The successful construction of pMTS7 provided a technical tool for studying mutants of mitochondrial genes in S.pombe.