金钱鱼孕激素受体基因的克隆、鉴定与表达研究

Molecular Cloning,Identification and Expression Analysis of Progesterone Receptor Gene in Spotted Scat Scatophagus argus

核受体(Pgr),隶属于核受体超家族,由核受体亚家族3C组成员3基因编码。孕酮通过结合Pgr介导其在脊椎动物繁殖过程中的生理学功能,因此,研究金钱鱼核受体基因(pgr基因)在不同组织及不同发育时期性腺中的表达模式,有助于为金钱鱼的繁殖提供理论依据。笔者克隆金钱鱼的pgr基因,用Megalign软件比较Pgr氨基酸同源性,用MEGA 7.0.4软件构建系统进化树,利用反转录PCR和实时荧光定量PCR分别探究pgr mRNA在金钱鱼各组织和性腺发育过程中的表达。金钱鱼pgr基因的开放阅读框(ORF)的长度为2016 bp,共编码671个氨基酸。氨基酸序列相似性分析发现,它与同属一个分支的鲈形目大黄鱼同源性最高。组织分布结果显示,pgr基因在雌、雄金钱鱼各组织中均有表达,且性腺、垂体中的表达最高。在垂体中,外源雌激素17β-雌二醇(E2)能显著升高pgr基因的表达。此外,随着性腺的发育,pgr基因在性成熟金钱鱼精巢、卵巢中的表达量最高。因此,Pgr对金钱鱼的生殖细胞发育成熟可能十分重要。

Progesterone receptor(Pgr)encoded by Nr3c3 belongs to the subfamily 3 group C of nuclear receptors.Progesterone was bind to its special target receptor Pgr to regulation its roles in production in vertebrates.In this study,progesterone receptor gene(pgr gene)of spotted scat Scatophagus argus was cloned.The RT-PCR and Real-time PCR were performed to explored the tissues distribution and expression of pgr mRNA in gonads at different stages,respectively.Comparison of pgr homology using Megalign software and construction of the phylogenetic tree using MEGA 7.0.4 software was performed in spotted scat.The pgr cDNA was identified and cloned.It was found that open reading frame(ORF)of pgr was 2016 bp,encoded 671 amino acids.Homologous sequence alignment analysis showed that pgr in large yellow croaker Larimichthys crocea displayed the higher similarity to that in spotted scat compared to other fish.RT-PCR analysis showed that pgr RNA was expressed in all tissues,with high expression in gonad and pituitary in which pgr mRNA expression was significantly up-regulated by exogenous E2(17β-Estrodiol).Furthermore,the expression of gonadal pgr mRNA was higher at maturation stage than in other developmental stage,which indicates that pgr plays an important role in regulation of the production of the matured eggs and sperm.