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

Abstract

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

CLC Number:

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.

References

[1] Nandi S,Raghu H M,Ravindranatha B M,et al.Production of buffalo (Bubalus bubalis) embryos in vitro:Premises and promises[J].Reprod Domest Anim,2002,37(2):65-74.
[2] Zicarelli L,Esposito L,Campanile G,et al.Effects of using vasectomized bulls in artificial insemination practice on the reproductive efficiency of Italian buffalo cows[J].Anim Reprod Sci,1997,47(3):171-180.
[3] 邓廷贤,庞春英,王建,等.水牛分子生物学技术研究新进展[J].中国畜牧兽医,2014,41(4):79-84.
[4] 崔奎青,张会娜,刘帅,等.沼泽型水牛NANOG基因5'调控序列克隆与功能分析[J].畜牧兽医学报,2013,44(3):387-394.
[5] 邓彦飞,刘真真,李云芳,等.不同逆转录载体系统应用于水牛胎儿成纤维细胞转基因的探索[J].华南农业大学学报,2013,4:558-563.
[6] 龚云,乔树叶,林浪,等.水牛YY1基因克隆及其shRNA片段的筛选[J].畜牧兽医学报,2014,45(1):62-68.
[7] 苏节,刘庆友,朱鹏,等.沼泽型水牛CYP19A1基因克隆、序列分析及组织表达研究[J].畜牧兽医学报,2013,44(4):514-521.
[8] Wu W,Han J,Cao R,et al.Sequence and regulation of the porcine FSHR gene promoter[J].Anim Reprod Sci,2015,154:95-104.
[9] 云无心.羊奶、水牛奶、骆驼奶——哪种奶"最好"[J].饮食科学,2012,2:10-11.
[10] Khan F A,Das G K,Pande M,et al.Alterations in follicular fluid estradiol,progesterone and insulin concentrations during ovarian acyclicity in water buffalo (Bubalus bubalis)[J].Anim Reprod Sci,2012,130(1-2):27-32.
[11] Perera B M.Reproductive cycles of buffalo[J].Anim Reprod Sci,2011,124(3-4):194-199.
[12] Di Francesco S,Novoa M V,Vecchio D,et al.Ovum pick-up and in vitro embryo production (OPU-IVEP) in Mediterranean Italian buffalo performed in different seasons[J].Theriogenology,2012,77(1):148-154.
[13] Pontes J,Sterza F M,Basso A,et al.Ovum pick up,in vitro embryo production,and pregnancy rates from a large-scale commercial program using Nelore cattle (Bos indicus) donors[J].Theriogenology,2011,75(9):1640-1646.
[14] Gromoll J,Pekel E,Nieschlag E.The structure and organization of the human follicle-stimulating hormone receptor (FSHR) gene[J].Genomics,1996,35(2):308-311.
[15] Gromoll J,Dankbar B,Gudermann T.Characterization of the 5' flanking region of the human follicle-stimulating hormone receptor gene[J].Mol Cell Endocrinol,1994,102(1-2):93-102.
[16] Heckert L L,Daggett M A,Chen J.Multiple promoter elements contribute to activity of the follicle-stimulating hormone receptor (FSHR) gene in testicular Sertoli cells[J].Mol Endocrinol,1998,12(10):1499-1512.
[17] Kim J S,Griswold M D.E2F and GATA-1 are required for the sertoli cell-specific promoter activity of the follicle-stimulating hormone receptor gene[J].J Androl,2001,22(4):629-639.
[18] 牛金涛,陆阳清,任子利,等.水牛促卵泡素受体(FSHR)基因5'端侧翼序列的克隆与分析[J].广西农业生物科学,2006,25(z1):198-199.
[19] Griswold M D,Kim J S.Site-specific methylation of the promoter alters deoxyribonucleic acid-protein interactions and prevents follicle-stimulating hormone receptor gene transcription[J].Biol Reprod,2001,64(2):602-610.
[20] Heckert L L,Griswold M D.The expression of the follicle-stimulating hormone receptor in spermatogenesis[J].Recent Prog Horm Res,2002,57:129-148.
[21] Wunsch A,Ahda Y,Banaz-Yasar F,et al.Single-nucleotide polymorphisms in the promoter region influence the expression of the human follicle-stimulating hormone receptor[J].Fertil Steril,2005,84(2):446-453.
[22] Cao Y,Zhao S J,Liang T Y,et al.Molecular cloning and functional analysis of the follicle-stimulating hormone (FSH) receptor gene promoter from the Jintang Black goat[J].African Journal of Biotechnology,2013,10(57):12118-12124.

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

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