酿酒酵母荧光定位报告菌株的开发

【目的】为了给外源蛋白在酿酒酵母细胞中的定位提供参考,构建酿酒酵母荧光定位报告菌株。【方法】运用染色体同源重组的方法,将突变的、已进行酵母表达优化的红色荧光蛋白RedStar分别整合到12个酵母细胞器标记蛋白的C端,与之进行融合表达,用特异性引物对每一个酵母荧光定位报告菌株进行PCR扩增和测序验证,用激光共聚焦显微镜进行荧光检测,对线粒体和细胞核进行特异性染料染色,用EGFP标记沙门氏菌已知定位蛋白SipA,与构建的相应荧光定位报告菌株进行共定位。【结果】构建的酿酒酵母荧光定位报告菌株可分别标示酵母细胞的肌动蛋白、晚期胞内体、细胞核、核周质、纺锤体、线粒体、过氧化物酶体、脂滴、初级内吞体、次级内吞体、高尔基体顺面及高尔基体反面。PCR扩增及测序验证、荧光检测、染料与相应报告菌株的共定位、已知定位蛋白SipA与相应报告菌株的共定位均提示报告菌株构建成功。【结论】这些报告菌株的构建,为日后在酵母中观察细胞器动态变化,以及未知蛋白在酵母中的定位提供了基础性工具。

Development of fluorescent localization reporter strains of the budding yeast

[Objective] To facilitate the localization of proteins in budding yeast, we developed yeast reporter strains expressing fusion proteins that localize to specific subcellular structures. [Methods] The yeast expression vectors expressed organelle-specific proteins tagged with much brighter variant red fluorescent protein (RedStar) at their carboxy terminal were constructed and transferred into yeast cells to generate the reporter strain collection. To this end, the yeast codon that optimized coding sequence of RedStar were in-frame inserted immediately preceding the stop codon of each ORF that controlled by their endogenous promoters. Each strain was verified by PCR and sequencing, and then analyzed by fluorescence microscopy. Co-localization of DNA-binding dyes (DAPI), Mitotracker (mitochondria) and SipA from Salmonella enteritidis in the corresponding reporter strain were also performed. [Results] The yeast strain collection containing indicators for the actin, endoplasmic reticulum, nuclei, mitochondria, peroxisomes, lipids, endosomes, and the Golgi apparatus were constructed successfully. [Conclusion] Our studies provide a fundamental tool for observing the dynamic changes of organelles and identifying the localization of unknown proteins in yeast.