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

作　　者：张亚伟[1] 安鹏[1] 张德锋[1] 刘明兰[1] 雷安民[1] 

机构地区：[1]西北农林科技大学动物医学院,陕西省干细胞工程技术研究中心,陕西省农业分子生物学重点实验室,杨凌712100

出　　处：《农业生物技术学报》2013年第3期320-327,共8页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金项目(No.31172280)

摘　　要：p53是一种肿瘤抑制基因,在细胞周期阻滞、细胞凋亡、衰老和自噬过程中发挥着调控作用,在机体的各个组织中广泛表达。本研究旨在构建牛p53基因真核表达载体并研究其在牛卵母细胞中的表达和定位。首先从牛(Bos taurus)卵丘细胞中克隆了p53基因并将其连接到pMD19-T载体上,双酶切鉴定后定向连接到pVenus载体上构建了pVenus-p53真核表达载体,经脂质体2000介导转染Hela细胞,确定重组质粒在Hela细胞中的表达定位,主要定位于细胞核。然后用体外转录试剂盒将p53-Venus转录成cRNA,通过显微注射观察其在卵母细胞中的表达定位情况,主要定位于细胞核,但胞质中也有少量表达。最后,取体外培养不同时期的牛卵母细胞,通过实时定量PCR检测p53在体外培养不同时期的表达量的变化,采用2-ΔΔCt法分析p53/β-actin表达水平的变化值,p53的表达从GⅤ期到MⅠ期迅速升高,在MⅠ期其表达量达到最高,随后又逐渐降低。本研究构建了牛p53真核表达载体pVenus-P53,并且体外转录的p53-VenuscRNA能在牛卵母细胞中正确表达定位,检测了p53在牛卵母细胞成熟培养各个时期表达量的变化,为进一步研究p53在牛卵母细胞体外成熟中的作用提供了参考资料。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 ofp53 gene in bovine oocyte. Firstly, we cloned the total CDs sequence ofp53 gene from bovine cumulus cells and linked the CDs sequence of bovine p53 gene into pMD19-T, then we identified pMD19-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 2a analysis to analyze the expression level ofp53/fl-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

关 键 词：P53基因 体外转录 显微注射 牛卵母细胞 

分 类 号：S823.3[农业科学—畜牧学]