半滑舌鳎(Cynoglossus semilaevis)gnrh2基因克隆、组织分布及卵巢成熟过程中表达分析

摘要

In order to clarify the physiological roles of gonadotropin-releasing hormone 2 (GnRH2) during ovarian maturation in half-smooth tongue sole (Cynoglossus semilaevis),we first cloned the full-length cDNA of GnRH2 by RT-PCR coupled with RACE.Then,the tissue distribution and changes in gnrh2 mRNA levels during ovarian maturation were evaluated by real-time quantitative PCR.Our data showed that the full-length cDNA of GnRH2 was 538 bp in size,excluding the poly-A tail.It consisted of a 5'untranslated region (UTR) of 154 bp,a 3'UTR of 126 bp and an open reading frame (ORF) of 258 bp which encoded an 85-amino acids preprohormone with a deduced molecular mass and isoelectric point of 9.69 kDa and 8.55,respectively.GnRH2 precursor contained a signal peptide,a mature decapeptide,a processing site,and a GnRH-associated peptide.Multiple sequence alignments indicated that GnRH2 preprohormones were highly conserved among teleosts,especially the decapeptide (QHWSHGWYPG) motif.Tongue sole GnRH2 precursor displayed the highest sequence identities with the order Perciformes (89.41％-90.59％),followed by Pleuronectiformes,Salmoniformes,and Tetraodontiformes (78.82％-85.88％),and the lowest sequence identities with Cypriniformes (61.18％-71.76％).The tissue distribution showed that gnrh2 transcripts could be detected at the highest levels in the brain and at lower levels in the pituitary and other peripheral tissues.The developmental stages of ovaries were divided into five stages (Ⅱ,Ⅲ,Ⅳ,Ⅴ,and Ⅵ) by histological analysis and the expression patterns of gnrh2 mRNA during ovarian maturation were also investigated.In the brain,the mRNA expression of gnrh2 peaked at stage Ⅲ,declined sharply at stage Ⅳ and reached a minimum at stage Ⅴ.However,the brain gnrh2 mRNA levels ascended again at stage Ⅴ.The pituitary gnrh2 mRNA levels declined gradually,except for a temporary increase at stage Ⅵ.These data indicate that brain GnRH2 appears to be involved in ovarian maturation in the half-smooth tongue sole,and this study extends our knowledge of the roles of GnRH2 on the regulation of reproduction in fish.%为了研究下丘脑神经肽促性腺激素释放激素(Gonadotropin-releasing hormone 2,GnRH2)在半滑舌鳎(Cynoglossus semilaevis)卵巢成熟过程中的生理作用,本研究通过RT-PCR及RACE方法获得了半滑舌鳎GnRH2全长cDNA序列;通过实时荧光定量PCR(qPCR)对gnrh2 mRNA的组织分布以及卵巢成熟过程中的时空表达特性进行了分析.结果显示,半滑舌鳎GnRH2全长cDNA序列为538 bp(不包括polyA尾),其中,5'非编码区(Untranslated region,UTR)为154 bp,3'UTR为126 bp,开放阅读框(Open reading frame,ORF)为258 bp,编码85个氨基酸的前体多肽,其分子量及等电点分别为9.69 kDa和8.55.GnRH2前体多肽由信号肽、GnRH2十肽、酶切位点(GKR)以及GnRH相关肽共4部分组成.序列比对分析发现,GnRH2在鱼类中同源性极高,尤其是十肽(QHWSHGWYPG)在所有硬骨鱼类中完全相同.半滑舌鳎GnRH2与鲈形目同源性最高(89.41％-90.5 9％),其次为鲽形目、鲑形目和鲍形目(78.82％-85.88％),与鲤形目同源性最低(61.18％-71.76％).gnrh2 mRNA主要在脑中表达,在垂体及其他外周组织中表达量极低.此外,组织学分析显示,半滑舌鳎卵巢发育共分为5个时期(Ⅱ、Ⅲ、Ⅳ、Ⅴ和Ⅵ期).在卵巢成熟过程中,脑gnrh2 mRNA表达量在卵黄生成期(Ⅲ期)显著性增加,达到峰值;随后表达量急剧下降,在成熟期(Ⅴ期)达到最小值;在排卵后期(Ⅵ期)又显著性增加.然而,在卵巢成熟过程中,垂体gnrh2 mRNA表达量在卵黄生成后期(Ⅳ期)显著性降低,随后在成熟期(Ⅴ期)有所增加,但在排卵后期(Ⅵ期)又急剧下降.上述研究结果表明,脑GnRH2可能参与了半滑舌鳎卵巢发育过程.