水牛促卵泡素受体(FSHR)基因5′端侧翼序列的克隆与分析

Cloning and analyzing the 5′region of follicle stimulating hormone receptor(FSHR) gene on buffalo

促卵泡素(FSH)是动物脑垂体分泌作用于卵巢卵泡生长、发育、分化、成熟和排卵的重要繁殖激素。由于是生物大分子,不能透过细胞膜,FSH必须与靶细胞膜上的促卵泡素受体(FSHR)结合,经过受体介导将信息传递到靶细胞内,才能发挥其生物学功能。目前国内外已对牛、山羊等多种动物的FSHR基因的cDNA序列进行了克隆测序,但是关于水牛FSHR基因5′端侧翼序列还未见报道。本研究对水牛FSHR基因5′端序列进行克隆和序列分析,其目的是寻找水牛FSH受体基因的SNP突变位点,从而为以后进行FSH受体基因的多态性分析和探索该受体基因在水牛繁殖中的可能作用打下基础。由水牛血液中提取DNA,与根据牛FSH受体基因序列设计的特定引物进行PCR反应,以合成FSHR 5′端侧翼调控区,扩增的该片断经分离、回收和纯化后再插入到pMD18-T质粒中,筛选阳性克隆,将插入了FSHR 5′端侧翼端片断的质粒纯化后进行序列测定和分析,弄清核苷酸序列和假定的氨基酸顺序并通过GeneBank进行同源性分析。结果表明,PCR扩增获得的片段为939 bp,与中国西门塔尔牛该段序列相比,同源性为97.44%,在-720 bp处的1个碱基缺失;与山羊的FSHR基因比较,同源性为94.78%,在-625^-619 bp处有7个碱基的插入;在此激素反应元件3′端一段序列(-530^-526 bp)水牛和山羊的相同均为TGACC,而西门塔尔牛为CGACC,但三者5′端一段序列(-679^-675 bp)没有突变并且相同,均为GGTCA;与人和大鼠FSHR基因不同的是,在牛、水牛和羊的FSHR基因启动子区检索出了TATA盒基元和多个类CAAT盒序列,且在水牛、山羊和牛之间没有碱基差异。因此,对水牛FSHR基因转录启动和表达调控值得进一步研究。

Follicle stimulating hormones （FSH）, which are secreted by pituitary, target to ovary and stimulate the growth,development,differentiation, maturation and ovulation of follicles. As a large biology molecular, FSHcan＇t penetrate the cell membranes of the target cells. In order exert their biology function, FSH have to bind to the follicle stimulating hormone receptors （FSHR） lying on the membrane of the target cells. At present, many domestic and foreign researchers have detected the sequences of FSHR on cattle, goat and so on. However, there＇s no report about the 5＇ region sequence of FSHR on buffalo. In this study, the 5＇ region sequence of this gene was cloned and analyzed and the comparison between the homology sequences of this gene on cattle and goat was also investigated. The aim of the study is to find the singlenueleotide polymorphism （SNP） mutation point in the buffalo FSHR gene and to set the base for further studies on its polymorphisms and the subsequent use of the gene on buffalo reproduction. The buffalo genome DNA was extracted from blood and the primers used for PCR were designed according to the bovineFSHR gene. AfterPCR amplification, the sequence was detected, recovered and purified. Then the sequence was inserted into pMD18-T vectors, and the positive clones were detected. The vectors carrying the target sequence were extracted and sequenced. The target sequence was also translated into amino acid sequence and homology was analyzed by GeneBank. The result showed that the PCR product of buffalo FSHR is 939 bp. Compared with the Chinese Simmental, the homology of this sequence is 97. 44% and there is 1 bp absence in--720 bp. Compared with goat, the homology of this sequence is 94.78% and there are 7 bp inserts between-- 625～ -- 619 bp. A part of sequence （--530～--526） in the 3＇ region of the hormone reaction element of buffalo is the same with that of goat, both of which are TGACC, but different from the Chinese Simmental, which was CGACC. However, the three species share the same sequence GGTCA in the 5＇ region （--679～--679 bp）. There are TATA box and severalCAAT boxqike sequences detected in the promoter region of FSHR gene in buffalo, goat and cattle and there is no difference among the three species, while there was no such boxes have been detected in human and rat yet. Further study should be carried out to illustrate the promoting and expressing regulation of buffalo FSHR gene.