绵羊AMH基因的克隆、序列分析与原核表达  

Molecular cloning,sequence analysis and prokaryotic expression of AMH gene of sheep

作　　者：蒋香菊[1,2] 吴阳升 林嘉鹏 汪立芹 张利[1,2] 古丽米热.阿不都热依木 杨楠[2,3] 刘莉[2,3] 黄俊成 

机构地区：[1]石河子大学动物科技学院,新疆石河子832000 [2]农业部草食家禽遗传育种与繁殖重点实验室,新疆乌鲁木齐830000 [3]新疆农业大学,新疆乌鲁木齐830000

出　　处：《江苏农业学报》2013年第6期1386-1392,共7页Jiangsu Journal of Agricultural Sciences

基　　金：国家自然科学基金项目(U1203381);自治区公益性科研院所基本科研业务经费项目;国家重大转基因专项(2013ZX08008-003);自治区科技支疆项目(201291147);自治区科技计划项目(201111113)

摘　　要：旨在克隆绵羊抗苗勒管激素基因(oAMH)的全长编码区序列,并对其序列进行分析。以绵羊的卵泡颗粒细胞全RNA逆转录得到的cDNA为模板,参照牛的AMH cDNA序列设计兼并引物,对oAMH基因的全长编码区进行T载体克隆并测序,并对其进行生物信息学分析。根据预测的编码区氨基末端260氨基酸对应的序列设计表达引物,构建原核表达载体pET15b-oamh260,转化BL21(DE3)菌,经IPTG诱导,对表达产物进行SDS-PAGE和Western blot鉴定。序列分析结果表明,获得的绵羊AMH基因序列全长1 797 bp(GenBank登录号:KC986978),完整开放阅读框为1 728 bp,共编码575个氨基酸,氨基末端24个氨基酸为信号肽序列,羧基末端区域TGFβ家族保守区,全序列与牛AMH的同源性为95.80%。SDS-PAGE结果显示,IPTG诱导表达的融合蛋白大小为30 000,与预期蛋白质大小一致。Western blot检测结果显示,该蛋白质为His融合蛋白。以上结果表明该试验成功克隆了绵羊AMH基因的完整编码区序列,构建了原核表达载体,在大肠杆菌中成功表达出目的融合蛋白,为进一步研究其生物学功能奠定了基础。Anti-Mullerian hormone （AMH）, also known as Mullerian inhibiting substance （MIS）, is a member of transforming growth factor β superfamily of growth and differentiation factors. In mouse, AMH participates in two critical selection points of follicle development, inhibiting the recruitment of primordial follicles into pool of growing follicles and decreasing the responsiveness of growing folli- cles to FSH（follicle-stimulating hormone）. However, little is known of the physiology of AMH in monovular species. This study aimed to obtain AMH full-length cDNA of mon- ovular species sheep and analyze the sequence. The tem- plate cDNA which was reversely transcribed from the total RNA of sheep granulosa cells was subjected to polymerase chain reaction （PCR） with the specific primers of AMH. The full- length eDNA of ovine AMH （oAMH） was obtained by cloning and sequencing. The N-terminal 260 amino acides encoded by ovine AMH were subeloned into the pET15b expession vector with Nde I/BamH I enzyme sites, and were transformed into BL21 （DE3） Escherichia coli. The induced products by IPTG（ isopropyl-13-d-thiogalaetoside） were analyzed by SDS-PAGE and Western blot. The oamh included 1 797 bp （ GenBank No. KC986978） with the open reading frame of 1 728 bp encoding 575 amino acides containing a 24 amino acid leader peptide. A comparison of sheep and other mammals AMH proteins re- vealed a highly conserved C-terminal domain that showed marked homology with transforming growth factor-13 family. The pre- dicted protein sequence shared 95.80% identity with cattle. SDS-PAGE analysis revealed that the expressed protein in BL21 （ DE3 ） was about 30 000. Western blot discovered that the expressed protein was a His tag fusion protein. Cloning and suc- cessful expression of ovine AMH would provide a foundation for further study on its biological function.

关 键 词：绵羊 AMH基因 序列分析 原核表达 

分 类 号：S826.2[农业科学—畜牧学]