绵羊FSHR基因可变剪接体的克隆、鉴定及表达分析

摘要

根据GenBank中绵羊促卵泡激素受体基因(FSHR)的mRNA序列(登录号:NM_001009289.1)设计引物,反转录PCR(RT-PCR)扩增蛋白编码区(CDs)区,以获取绵羊FSHR基因可变剪接体,并通过Western blot验证其在组织中的表达,通过免疫组织化学方法检测其在卵泡中的表达位置.结果表明,在绵羊卵巢中除了全长的、编码695个氨基酸的FSHR表达产物外,还鉴定到分别编码694个、633个、595个、582个和533个氨基酸的FSHR可变剪接形式的转录产物.在卵泡中主要以695(694)、633和5953种转录本形式存在.但在颗粒细胞中绵羊促卵泡激素受体主要以经典分子即695个氨基酸的形式存在.在卵泡发育过程中,FSHR蛋白质含量发生变化:在初级卵泡中,FSHR主要在卵母细胞中表达,而不是在单层的颗粒细胞中表达,在次级卵泡中,FSHR在颗粒细胞和卵母细胞中均不表达,到三级卵泡(有腔卵泡)时,FSHR蛋白质在颗粒细胞中表达丰富.可见,绵羊FSHR基因虽然存在可变剪接体的结构,但其功能主要取决于经典型的695个氨基酸蛋白质分子,并且对初级卵泡的发育具有一定的作用.%Based on the sequence of FSHR gene in GenBank ( accession number:NM_001009289.1) , primers were designed, and the coding sequence ( CDs) of ovine FSHR were obtained by reverse transcription-polymerase chain reaction (RT-PCR). Temporal and spatial expression patterns were assessed by western blot and immunohistochemistry (IHC) in different tissues and follicles of sheep. The results showed that, in addition to a transcript encoding the classical 695 amino acids( aa) , there are more than six FSHR splicing variants encoding proteins of 694 aa, 670 aa, 633 aa, 595 aa, 582 aa and 533 aa in sheep granulosa cells. Three transcripts (695/694 aa, 633 aa and 595 aa) were found in the follicles with different diameters. However, only the classical protein is translated in granulosa cells. In primary follicles, FSHR proteins were abundant in oocytes but not in monolayer granulosa cells. In secondary follicles, FSHR proteins were downregulated, both in granulosa cells and oocytes. In tertiary ( antral) follicles, FSHR proteins were abundant in granulosa cells. It was indicated that sheep FSHR plays a role in the development of primary follicles despite of the existance of FSHR alternative splicing isoforms, and it' s function depends on the classical FSHR protein.