条石鲷促性腺激素α亚基的克隆与表达特性分析

Cloning and Expression Pattern of Gonadotropin Hormone Common-alpha Subunit in Oplegnathus fasciatus

采用同源性克隆和cDNA末端快速扩增（RACE）方法,从条石鲷（Oplegnathidae fasciatus）垂体中获得了全长为897bp的GtHαcDNA序列,其中包含249bp的5′非编码区、399bp的开放阅读框和249bp的3′非编码区。该基因编码由132个氨基酸组成的前体蛋白,其中前38个氨基酸为信号肽,在第122和124Cys中间间隔了一个组氨酸（His）,形成CXC型趋化因子的特征结构,肽链中包括10个保守Cys残基和2个N-糖基化位点。氨基酸同源性和进化分析表明,条石鲷GtHα与鲈形目鱼类的进化关系较近,与鲈形目鱼类GtHα的同源性为87%-98%,与其他脊椎动物GtHα的同源性为53%-87%。实时荧光定量PCR检测发现,GtHαmRNA在垂体、脑、性腺中的表达量较高,在心、肝脏、胃、肠、肌肉、幽门盲囊中微量表达,在脾脏、头肾、鳃、肾脏中不表达,其中在垂体中的表达量最高;周期表达发现：垂体中GtHαmRNA在卵巢发育的Ⅴ期达到最大值,卵巢中GtHαmRNA在卵巢发育的Ⅳ期达到最大值,而脑组织中GtHαmRNA在卵巢发育的Ⅴ期达到最低值,表明GtHα对卵母细胞发育的调控可能同时通过自分泌和旁分泌两种途径进行。本研究为条石鲷生殖调控机制及人工繁育技术研究提供了基础资料。

A full length cDNA sequence of gonadotropin hormone common alpha subunit was cloned from the pituitary of Oplegnathus fasciatususing by homology cloning and RACE techniques. The GtHα cDNA sequence consisted of 897 base pairs nueleotides, including 249 base pairs at 5＇ untranslation region, 249 base pairs at 3r untranslation region and an open reading frame with 399 base pairs nucleotides. The cDNA sequence encoded a precursor protein of 132 amino acids preceded by a signal peptide of 38 amino acids residues. The putative peptide contained ten cysteine residues and two N-glycosylation sites residues. A histidine residue in the middle of 122 and 124 cysteine formed a CXC chemokine characteristic structure. Sequences comparison and phylogenetic analysis showed that the GtHα of Oplegnathus fasciatus had higher identity to their orthologs of Perciformes （87%-98%） than to other vertebrates （53%-87 % ）. The qRTPCR results suggested that the level of GtHα mRNA was higher in pituitary, ovary, brain and trace expression in heart, liver, stomach, intestine, muscle and pyloric caecum. However, it was not found in kidney, Head kidney, spleen and gill by Real-time Quantitative PCR technique. The level of GtHα mRNA was highest in pituitary. The reproductive cycle expression showed that the level of GtHα mRNA in pituitary was maximum in the stage V of ovarian development and that in ovary was maximum in the stage Ⅳ. However, the level in brain was lowest in the stage V suggesting the regulation of GtHα in the ovarian development may through both autocrine and paracrine pathways. This research provides a reference to the reproductive regulation mechanism and artificial breeding technigues of Opiegnathus fasciatus.