水牛Smad4基因的克隆及其真核表达载体的构建

doi:10.16431/j.cnki.1671-7236.2017.08.022

《中国畜牧兽医》 ›› 2017, Vol. 44 ›› Issue (8): 2369-2377.doi: 10.16431/j.cnki.1671-7236.2017.08.022

水牛Smad4基因的克隆及其真核表达载体的构建

鄢胜飞, 张秀芳, 黄玥萌, 郑海英, 杨春艳, 覃广胜, 黄加祥, 尚江华

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:2017-03-15 出版日期:2017-08-20 发布日期:2017-08-18
通讯作者: 郑海英,尚江华 E-mail:haiyingzheng@126.com;jh-shang@tom.com
作者简介:鄢胜飞(1989-),男,湖北天门人,硕士,研究实习员,研究方向:动物胚胎发育,E-mail:yanshengfei2015@163.com
基金资助:
广西科技计划项目（桂科AB16380040）；广西自然科学基金（2014GXNSFAA118116）；广西水产畜牧兽医局科技项目（桂渔牧科201633052）；广西水牛研究所基本科研业务费（水牛基1404004、160204、1705001）

Cloning and Construction of Eukaryotic Expression Vector of Buffalo Smad4 Gene

YAN Sheng-fei, ZHANG Xiu-fang, HUANG Yue-meng, ZHENG Hai-ying, YANG Chun-yan, QIN Guang-sheng, HUANG Jia-xiang, SHANG Jiang-hua

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

Received:2017-03-15 Online:2017-08-20 Published:2017-08-18

摘要/Abstract

摘要：

为了阐明Smad4基因在水牛卵巢颗粒细胞增殖及胚胎发育中的分子机制，试验采用RT-PCR扩增并克隆水牛Smad4基因，对其核苷酸序列和蛋白质序列进行生物信息学分析，构建Smad4基因的真核表达载体，通过脂质体转染法转染体外培养的牛卵巢颗粒细胞。结果表明，试验克隆得到水牛Smad4基因编码序列，编码区全长为1 662 bp，编码553个氨基酸。通过BLAST对水牛Smad4基因的核苷酸序列进行同源性比对，结果显示，水牛Smad4基因与牛的同源性为99%，与绵羊、猪、马、人的同源性分别为98%、96%、96%和95%。系统进化树分析表明，Smad4基因在不同物种的进化过程中具有高度的保守性。试验成功构建了水牛Smad4基因的真核表达载体pEGFP-N1-Smad4，并在水牛卵巢颗粒细胞中表达出较强的绿色荧光蛋白Smad4-EGFP融合蛋白。本研究成功克隆了水牛Smad4基因，并成功构建了Smad4基因的真核表达载体，为进一步研究Smad4基因在水牛胚胎发育过程中的分子机制奠定基础。

关键词: 水牛; Smad4基因; 克隆; 真核表达载体

Abstract:

This study was aimed to elucidate the molecular mecbanisms of Smad4 gene on granulose cells proliferation and embryonic development in buffalo. The Smad4 gene was amplified by RT-PCR, analyzed by bioinformatics, studied with eukaryotic vector construction, and used liposome transfection skill to transfect into the buffalo granulose cells. The results showed that Smad4 gene was cloned, the coding region was 1 662 bp, and encoded 553 amino acids. BLAST analysis showed that the buffalo Smad4 gene shared 99% of similar nucleotide sequence with Bos taurus, and shared 98%, 96%, 96% and 95% of similar nucleotide sequence with Ovis aries, Sus scrofa, Equues caballus and Homo sapiens, respectively. Phylogenetic tree analysis showed that Smad4 gene was highly conserved in different species. The buffalo pEGFP-N1-Smad4 eukaryotic expression vector was successfully constructed, and transferred into the buffalo granulose cells, and the Smad4-EGFP fusion protein was detected in the cells. The results suggested that the success cloning and construction vector of buffalo Smad4 gene laid the foundation for the research of Smad4 gene on embryo development.

Key words: buffalo; Smad4 gene; cloning; eukaryotic expression vector

中图分类号:

Q785

鄢胜飞, 张秀芳, 黄玥萌, 郑海英, 杨春艳, 覃广胜, 黄加祥, 尚江华. 水牛Smad4基因的克隆及其真核表达载体的构建[J]. 《中国畜牧兽医》, 2017, 44(8): 2369-2377.

YAN Sheng-fei, ZHANG Xiu-fang, HUANG Yue-meng, ZHENG Hai-ying, YANG Chun-yan, QIN Guang-sheng, HUANG Jia-xiang, SHANG Jiang-hua. Cloning and Construction of Eukaryotic Expression Vector of Buffalo Smad4 Gene[J]. , 2017, 44(8): 2369-2377.

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水牛Smad4基因的克隆及其真核表达载体的构建

Cloning and Construction of Eukaryotic Expression Vector of Buffalo Smad4 Gene

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