农业生物技术学报2013，Vol.21Issue(3)：320-327,8.DOI:10.3969/j.issn.1674-7968.2013.03.009
牛p53基因真核表达载体构建及其在卵母细胞中表达定位
Construction of Eukaryotic Expression Vector pVenus-p53 and Its Expression and Localization in the Bovine (Bos taurus) Oocyte
摘要
Abstract
p53 is a kind of tumor suppressor genes, it plays a role in the process of controlling cell cycle arrest, senescence, autophagy and apoptosis, and it was espressed widely in various tissues of organism. The objectives of this study were to construct a eukaryotic expression vector pVenus-p53 and research the expression and localization of p53 gene in bovine oocyte. Firstly, we cloned the total CDs sequence of p53 gene from bovine cumulus cells and linked the CDs sequence of bovine p53 gene into pMD19-T, then we identified pMD\9-T-p53 by double restriction enzyme digestion and cloned the p53 into expression vector pVenus, and then we constructed eukaryotic expression vector of pVenus-p53. After pVenus-p53 recombinant plasmid transfected into Hela cells mediated by Lipofectamine 2000, we confirmed the expression and position of the recombinant plasmid pVenus-p53 in Hela cells. We found that pVenus-p53 recombinant plasmid mainly located in the nucleus of the Hela cells. Then p53-Venus was transcribed into cRNA using in vitro transcription kit. After that, we observed the expression and localization of p53-Venus cRNA in bovine oocyte after microinjection. We found that it expressed not only in cell nucleus, but also in cytoplasm, but the expression amount was much smaller than that in nucleus. Finally, we collected bovine oocytes in different developmental stages in vitro culture, and detected the changes of p53 mRNA expression in different developmental stages in vitro culture via the Real-time PCR. We used 2-ΔΔCt analysis to analyze the expression level of p53/β-actin and the result showed that the expression of p53 mRNA lifted quickly from G V stage to M Ⅰ period and the expression quantity was the highest in M Ⅰ period, and then it gradually decreased. In this study we constructed the p53 eukaryotic expression vector pVenus-p53 and the p53-Venus cRNA which could be expressed and localized correctly in bovine oocyte. We also detected the changes of p53 mRNA expression in different developmental stages of bovine oocyte in vitro culture. Our study provides reference material for further research in the role of p53 gene in bovine oocyte in vitro maturation.关键词
p53基因/体外转录/显微注射/牛卵母细胞Key words
p53 gene/ in vitro transcription/ Microinjection/ Bovine oocytes引用本文复制引用
张亚伟,安鹏,张德锋,刘明兰,雷安民..牛p53基因真核表达载体构建及其在卵母细胞中表达定位[J].农业生物技术学报,2013,21(3):320-327,8.基金项目
国家自然科学基金项目(No.31172280) （No.31172280）
