水牛STAT4基因克隆分析及其真核表达载体的构建

doi:10.16431/j.cnki.1671-7236.2016.08.001

《中国畜牧兽医》 ›› 2016, Vol. 43 ›› Issue (8): 1929-1937.doi: 10.16431/j.cnki.1671-7236.2016.08.001

• 生物技术 • 下一篇

水牛STAT4基因克隆分析及其真核表达载体的构建

朱鹏, 庞春英, 段安琴, 邓廷贤, 陆杏蓉, 梁贤威

中国农业科学院广西水牛研究所, 广西水牛遗传繁育重点实验室, 南宁 530001

收稿日期:2016-01-21 出版日期:2016-08-20 发布日期:2016-08-23
通讯作者: 梁贤威 E-mail:liangbri@126.com
作者简介:朱鹏(1986-),男,江西高安人,博士,助理研究员,研究方向:动物生殖生理,E-mail:yijianrudi@163.com
基金资助:
农业部转基因重点项目（2014ZX0801012B）；广西国合桂科合（14123001-5）；水牛人才（1502）

Cloning and Construction of Eukaryotic Expression Vector of Buffalo STAT4 Gene

ZHU Peng, PANG Chun-ying, DUAN An-qin, DENG Ting-xian, LU Xing-rong, LIANG Xian-wei

Guangxi Key Laboratory of Buffalo Genetics, Breeding and Reproduction, Guangxi Buffalo Research Institute, Chinese Academy of Agricultural Sciences, Nanning 530001, China

Received:2016-01-21 Online:2016-08-20 Published:2016-08-23

摘要/Abstract

摘要：

为了阐明水牛信号转导子和转录激活子4（signal transducer and activator of transcription 4, STAT4）基因在水牛卵泡、胚胎发生及泌乳调节过程中的作用及分子机制，本试验采用了3’-RACE克隆获得STAT4基因，并对其核苷酸序列和蛋白质序列进行了信息学分析，通过构建其真核表达载体并转染HEK293T细胞验证所构建载体的准确性。结果表明，水牛STAT4基因编码区长2 247 bp，3’-UTR区长268 bp，编码748个氨基酸。BLAST分析显示，水牛STAT4核苷酸序列与牛、山羊、绵羊、猪、马、犬和人相应序列的同源性分别为99％、99％、99%、95％、93％、93％和92％，系统进化树分析结果表明，STAT4基因在不同物种及进化的过程中具有高度保守性。蛋白质分析结果表明，STAT4蛋白呈弱酸性，无信号肽，定位于细胞质，存在STAT_int、STAT_alpha、STAT_bind和SH2_STAT4等结构域。microRNA靶标预测显示bta-miR-200a、bta-miR-2429和bta-miR-2410等为STAT4 3’-UTR潜在microRNAs。试验成功构建了水牛STAT4基因真核表达载体pEGFPN1-STAT4，转染HEK293T后产生较强的绿色荧光信号，表明能够形成STAT4-EGFP融合蛋白。

关键词: 水牛; STAT4基因; 克隆; 生物信息学分析; 载体构建

Abstract:

In order to elucidate the function and molecular mechanisms of buffalo signal transducer and activator of transcription 4 (STAT4) gene during folliculogenesis,embryogenesis and lactogenesis,buffalo STAT4 gene was studied with 3' -RACE,bio-informatics analysis,eukaryotic vector construction and cell transfection technologyin this study.The results showed that the coding region of buffalo STAT4 was 2 247 bp,3'-was 268 bp,and encoded 748 amino acids;BLAST analysis showed that the buffalo STAT4 gene shared 99%,99%,99%,95%,93%,93% and 92% of similar nucleotide sequence with that of Bos taurus, Capra hircus,Ovis aries,Sus scrofa,Equus caballus,Canis lupus and Homo sapiens,respectively.STAT4 protein was weakly acidic,without signal peptide,located in the cytoplasmic,and with the presence of STAT_int,STAT_alpha,STAT_bind and SH2_STAT4 domain.bta-miR-200a,bta-miR-2429 and bta-miR-2410 and so on were potential microRNAs of STAT4 3'-UTR.The buffalo STAT4 eukaryotic expression vector was successfully constructed,after transfected into HEK293T cell lines,STAT4-EGFP fusion protein was detectable.

Key words: buffalo; STAT4 gene; cloning; bio-informatics analysis; vector construction

中图分类号:

朱鹏, 庞春英, 段安琴, 邓廷贤, 陆杏蓉, 梁贤威. 水牛STAT4基因克隆分析及其真核表达载体的构建[J]. 《中国畜牧兽医》, 2016, 43(8): 1929-1937.

ZHU Peng, PANG Chun-ying, DUAN An-qin, DENG Ting-xian, LU Xing-rong, LIANG Xian-wei. Cloning and Construction of Eukaryotic Expression Vector of Buffalo STAT4 Gene[J]. , 2016, 43(8): 1929-1937.

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水牛STAT4基因克隆分析及其真核表达载体的构建

Cloning and Construction of Eukaryotic Expression Vector of Buffalo STAT4 Gene

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