水牛促卵泡素受体(FSHR)基因启动子克隆、生物信息学分析及转录活性检测

doi:10.16431/j.cnki.1671-7236.2015.11.003

›› 2015, Vol. 42 ›› Issue (11): 2833-2842.doi: 10.16431/j.cnki.1671-7236.2015.11.003

水牛促卵泡素受体(FSHR)基因启动子克隆、生物信息学分析及转录活性检测

朱鹏, 庞春英, 邓廷贤, 段安琴, 陆杏蓉, 陈明棠, 杨炳壮, 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:2015-04-03 出版日期:2015-11-20 发布日期:2015-11-26
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,博士生,研究方向:动物繁殖,E-mail:yijianrudi@163.com
基金资助:
农业部转基因重点项目(2014ZX08010-012B);广西水产畜牧局科技推广应用桂渔牧科(201452032);水牛基(1504006)

Cloning,Bioinformatics Analysis and Transcriptional Activity Detection of Buffalo FSHR Gene Promoter

ZHU Peng, PANG Chun-ying, DENG Ting-xian, DUAN An-qin, LU Xing-rong, CHEN Ming-tang, YANG Bing-zhuang, 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:2015-04-03 Online:2015-11-20 Published:2015-11-26

摘要/Abstract

摘要： 试验旨在对广西本地水牛FSHR基因5'侧翼序列进行克隆、生物信息学分析及其转录活性检测。根据GenBank已公布的黄牛FSHR 5'侧翼序列,本试验设计引物,以广西本地水牛血液基因组为模板扩增FSHR基因5'侧翼序列并进行生物信息学分析。结果显示本试验成功克隆了广西本地沼泽型水牛FSHR 5'侧翼序列及部分CDS区序列,共2979 bp,同源性比对分析结果表明其与河流型水牛、黄牛、绵羊、山羊、猪和人的同源性分别为100%、99%、93%、92%、91%和75%。对其5'侧翼2000 bp序列进行启动子预测及转录因子结合位点预测,结果显示在其翻译起始位点上游-147 bp附近存在TATA box,启动子区存在GATAs、FOXO1、FOXO3、Nobox、STAT1、STAT3、STAT4、STAT5A、STAT5B、STAT6和YY1等反式作用元件结合位点,其中GATAs家族基因在FSHR启动子区存在多个结合位点,且同一位点又存在多个GATAs家族基因结合的情况。水牛FSHR启动子能启动EGFP在HEK-293T细胞系中的表达,但表达非常微弱;也能启动EGFP在CHO细胞系中表达,且与CMV启动EGFP在CHO细胞系中的强度相似,结果表明水牛FSHR是个强启动子。总之,本研究成功克隆了沼泽型水牛FSHR基因启动子,分析了其启动子序列特征并成功验证其组织特异性的转录活性,为后期水牛繁殖性能分子机理阐明及基于卵巢特异性表达外源基因的转基因水牛奠定了基础。

关键词: 水牛; FSHR; 克隆; 生物信息学分析; 转录活性

Abstract: In this study,the 5'flanking sequence of FSHR gene was cloned,and bioinformatics analysis and transcriptional activity detection were studied.One pair of primers was designed among the homologous of 5'flanking sequence of FSHR gene in different species,5'flanking sequence of FSHR gene was cloned by PCR and constructed into the EGFP reporter vector,the transcriptional activity of recombinant expression vector pFSHR-EGFP-1 was analyzed by transfecting into HEK-293T and CHO cell lines.The results showed that 2979 bp of 5'flanking sequence of FSHR gene and partial CDS region of Guangxi local swamp buffalo was successfully cloned and sequenced.The homology comparison result showed that buffalo FSHR gene shared 100%,99%,93%,92%,91% and 75% homologies of nucleotide sequence with that of the river type buffalo,cattle,sheep,goat,pig and human,respectively.Promoter and transcription factor binding site predictions showed that there were a TATA box in the -147 bp area of the upstream of translation initiation site,and trans acting element binding sites for GATAs,FOXO1,FOXO3,Nobox,STAT1,STAT3,STAT4,STAT5a,STAT5b,STAT6 and YY1.There were multiple binding sites for GATAs family genes in the FSHR gene promoter region,and in the same site of FSHR promoter,it displayed multiple GATAs bound.The FSHR gene promoter could initiate EGFP expression in HEK-293T cell lines,however very weakly,the expression of EGFP promoted by FSHR promoter was similar with by CMV promoter in CHO cells lines,and suggested that buffalo FSHR was a strong promoter.In conclusion,this study had successfully cloned FSHR gene promoter,analyzed its promoter sequence characteristics and successfully verified the tissue specific transcriptional activity.It laid the foundation for clarifying the transcriptional regulatory mechanism of buffalo FSHR gene and produced transgenic buffalo base on ovary specific expression of exogenous gene.

Key words: buffalo; FSHR; clone; bioinformatics analysis; transcriptional activity

中图分类号:

朱鹏, 庞春英, 邓廷贤, 段安琴, 陆杏蓉, 陈明棠, 杨炳壮, 梁贤威. 水牛促卵泡素受体(FSHR)基因启动子克隆、生物信息学分析及转录活性检测[J]. , 2015, 42(11): 2833-2842.

ZHU Peng, PANG Chun-ying, DENG Ting-xian, DUAN An-qin, LU Xing-rong, CHEN Ming-tang, YANG Bing-zhuang, LIANG Xian-wei. Cloning,Bioinformatics Analysis and Transcriptional Activity Detection of Buffalo FSHR Gene Promoter[J]. , 2015, 42(11): 2833-2842.

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水牛促卵泡素受体(FSHR)基因启动子克隆、生物信息学分析及转录活性检测

Cloning,Bioinformatics Analysis and Transcriptional Activity Detection of Buffalo FSHR Gene Promoter

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