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目的:探讨小鼠GV期卵母细胞线粒体中ATP合酶亚基8(ATP8)基因的表达情况及其生物信息学的分析。方法:应用挤压法从卵巢中分离获得生发泡期(germinal vesicle,GV)卵母细胞,用RT-PCR检测GV期单个卵母细胞中ATP8基因的表达;将GV期单个卵母细胞直接进行RT合成cDNA,然后再对回收产物构建克隆质粒并测序,最后用VECTOR NIT9.0、RNAdraw和Treeview等工具对测序结果进行生物信息学分析。结果:琼脂糖电泳结果表明ATP8基因在GV期卵母细胞中有表达;测序结果显示所测昆明小鼠ATP8序列与Genbank序列完全一致;mRNA二级结构图显示12种动物间的ATP8氨基酸序列和ATP8核酸序列有较大差异,其中不同品种小鼠间也有差异;不同物种ATP8核苷酸序列构建出的分子进化树中,小鼠与棕熊进化距离较远,与田鼠、家猫进化距离相对较近。结论:小鼠GV期卵母细胞特异表达的ATP8基因与卵母细胞的正常发育成熟相关。
Objective: To investigate the expression of ATP synthase subunit 8 (ATP8) gene in mitochondria of mouse GV oocyte and its bioinformatics analysis. Methods: The oocyte was isolated from the ovary by extrusion method. The expression of ATP8 gene was detected by RT-PCR in a single oocyte of GV stage. The single oocyte in GV stage was directly treated RT. The cloned plasmids were constructed and sequenced. Finally, the sequencing results were analyzed by bioinformatics using tools such as VECTOR NIT9.0, RNAdraw and Treeview. Results: The results of agarose gel electrophoresis showed that the ATP8 gene was expressed in GV oocytes. The sequencing results showed that the ATP8 sequence of Kunming mouse was exactly the same as that of Genbank. The secondary structure of mRNA showed ATP8 amino acid sequence of 12 species ATP8 nucleotide sequences have great differences, of which there are differences between different species of mice; ATP8 nucleotide sequences of different species constructed phylogenetic tree, mice and brown bear evolutionary distance farther, and Microtus mandarinus than domestic cat evolutionary distance near. Conclusion: The ATP8 gene specifically expressed in mouse GV oocytes is related to the normal maturation of oocytes.