水牛p21基因在卵母细胞体外成熟过程中的表达及其真核表达载体构建  

Expression of p21 Gene in Water Buffalo Oocytes during in vitro Maturation and Construction of Its Eukaryotic Expression Vector

作　　者：郑海英[1] 黄玥萌 杨春艳[1] 鄢胜飞 李孟琪 于农淇 郑威[1] 黄加祥[1] 尚江华[1] ZHENG Hai-ying;HUANG Yue-meng;YANG Chun-yan;YAN Sheng-fei;LI Meng-qi;YU Nong-qi;ZHENG Wei;HUANG Jia-xiang;SHANG Jiang-hua(Guangxi Buffalo Research Institute,Chinese Academy of Agricultural Sciences,Nanning,Guangxi,530001,China)

机构地区：[1]中国农业科学院广西水牛研究所/农业部(广西)水牛遗传繁育重点试验室,广西南宁530001

出　　处：《动物医学进展》2018年第10期18-24,共7页Progress In Veterinary Medicine

基　　金：广西科技计划项目(桂科ABl6380040);广西自然科学基金项目(2014GXNSFAA118116);广西水产畜牧兽医局科技项目(桂渔牧科201528015,201528016,201633014);广西水牛研究所基本科研业务费(水牛基160205)

摘　　要：旨在检测p21基因在水牛卵母细胞体外成熟过程中的表达变化,并克隆摩拉水牛p21基因CDs序列,构建真核表达载体。收集体外成熟培养不同时期(0、6、12、24h)和不同形态[有第1极体(first polar body,PB1)PB1和无PB1]的水牛卵母细胞,通过RT-qPCR检测其p21mRNA相对表达量。同时利用RTPCR技术从摩拉水牛卵巢颗粒细胞中扩增出p21基因编码序列,插入pEGFP-N1真核表达载体中,构建出重组质粒。将重组质粒pEGFP-N1-p21转染水牛颗粒细胞,并于培养24h和48h后观察转染细胞的荧光表达情况。收集转染48h后的颗粒细胞,通过RT-qPCR检测p21mRNA相对表达量。结果显示,成熟培养6h的卵母细胞p21mRNA表达量显著高于成熟培养0、12、24h的表达量(P<0.05),有PB1的卵母细胞中p21mRNA的表达量显著高于无PB1的卵母细胞(P<0.05);重组质粒pEGFP-N1-p21转染颗粒细胞后,有绿色荧光蛋白表达,且与转染pEGFP-N1和空白组相比,pEGFP-N1-p21转染组的p21mRNA表达量显著升高。结果表明,在体外成熟培养过程中均能检测到水牛卵母细胞p21mRNA表达量,成功构建了摩拉水牛p21基因真核表达载体,为下一步研究p21基因在水牛卵母细胞成熟和胚胎发育过程中的功能和调控奠定了基础。The objective of this study was to determine the expression of p21 gene of buffalo oocytes during IVM and to clone the CDs of p21 gene to construct the eukaryotic expression vector of Murrah buffalo.The buffalo oocytes of different periods （0,6,12,24 h） and different morphologies （with PB1 and no PB1） werecollected during in vitro oocyte maturation （IVM）,and the expression of p21 gene was detected by qRT- PCR.The coding sequence （CDs） of p21 gene was amplified from the ovarian granulosa cells of Murrah buffalo using RT-PCR technique.The p21 sequence was inserted into the pEGFP-N1 to construct the pEG- FP-NI-p21 eukaryotic expression vector.The plasmids were transfected into ovarian granulosa cells and in- vestigated by the green fluorescence under microscope after 24 h and 48 h.The relative transcription levels of p21 gene mRNA were determined by qRT-PCR 48h after transfecting ovarian granulosa cells.The results showed that the expression of p21 gene in 6h during IVM was significantly increased compared with other groups （P-0.05）,and the expression of p21 gene in oocytes that emitted the first polar body was signifi- cantly higher than those oocytes that did not （P%0.05） - The pEGFP-Nl-p21 vector was constructed suc- cessfully and the green fluorescence was observed in the transfected cells.Compared with pEGFP-N1 ,NC, the relative level of p21 gene mRNA increased significantly in ovarian granulosa cells which transfected with pEGFP-Nl-p21.In conclusion, these data demonstrated that expression of p21 gene was detected in different phases of buffalo ooctyes,and the pEGFP-N1-p21 was constructed successfully.This greatly facili- tated the further study of function and regulation of p21 gene,especially its role on oocytes maturation and early embryo development in buffalo.

关 键 词：摩拉水牛 P21基因表达 颗粒细胞 pEGFP-N1载体 

分 类 号：S858.23[农业科学—临床兽医学] Q786[农业科学—兽医学]