粟酒裂殖酵母中Mef2在线粒体中功能的研究

摘要

Mitochondrial translation,essential for synthesis of the electron transport chain complexes in the mitochondria,is governed by nuclear encoded gene. This paper aims to reveal the main function of Mef2 protein in Schizosaccharomyces pombe. First,we constructed the strain of mef2 gene deletion by homologous recombination,and observed its growth phenotype in non-fermentative medium with glycerol as the sole carbon source. Second,the bioinformatics analysis demonstrated that the N-terminal of Mef2 contained a mitochondrial localization sequence(MTS)consisting of 31 amino acids. To further determine the localization of Mef2 protein,a GFP fluorescent label was added to the C-terminus of Mef2 to observe GFP green fluorescence. Third,Northern blotting was used to detect the effect of mef2 deletion on the level of mitochondrial genome encoding mRNAs. Finally,Western blotting was employed to detect the influence of the deletion of mef2 on the expression of mitochondrial genome-encoded protein. The results showed that Δmef2 strain caused growth defects on non-fermentation medium, which was one with mitochondrial respiratory defect. GFP green fluorescence localization experiments confirmed that Mef2 was localized in mitochondria. Northern blotting revealed that the deletion of mef2 did not affect the transcription of mRNAs encoded by the mitochondrial genome. Western blotting showed that the deletion of mef2 resulted in decrease in the expression of Cox1,Cox3,Atp6,and Cob1 proteins. In summary,Mef2 is a protein that is closely related to mitochondrial function and is involved in the translation of mitochondrial encoded proteins Cox1,Cox3,Atp6,and Cob1.%由核编码基因控制的线粒体翻译对线粒体中电子传递链复合物的合成是必不可少的.旨在揭示粟酒裂殖酵母中Mef2蛋白的主要功能.利用同源重组的方法构建 Δmef2突变体,观察 Δmef2在以甘油为唯一碳源的非发酵培养基的生长表型;生物信息学分析结果显示Mef2的N端含有一段由31个氨基酸组成的线粒体定位序列(MTS),为进一步确定Mef2蛋白的定位,在Mef2的C端添加一个GFP荧光标记,观察GFP绿色荧光的位置.接着采用Northern blotting检测mef2的缺失对线粒体基因组编码mRNAs的影响.最后,运用Western blotting检测mef2的缺失对线粒体基因组编码的蛋白的影响.研究结果表明,Δmef2菌株在非发酵培养基上表现出生长缺陷,是线粒体呼吸缺陷型菌;GFP绿色荧光定位实验证实了Mef2定位于线粒体中;Northern blotting实验结果显示mef2的缺失不影响线粒体基因组编码mRNAs的转录;Western blotting检测结果显示mef2的缺失导致Cox1、Cox3、Atp6和Cob1蛋白的表达量降低.综上所述,Mef2是一个与线粒体功能密切相关的蛋白,并且参与了线粒体编码蛋白Cox1、Cox3、Atp6和Cob1的翻译.