泥蚶胰岛素生长因子1受体基因的cDNA全长克隆及表达分析

为了研究IGF1R基因在泥蚶生长、发育过程中的调控作用,本研究基于泥蚶转录组文库的EST,利用SMART RACE技术首次克隆获得泥蚶IGF1R基因(Tg-IGF1R)c DNA全长序列,该序列全长为5793 bp,开放阅读框为4638 bp,编码1546个氨基酸。经生物信息学分析发现,该基因5′端非编码区为810 bp,3′端非编码区为345 bp,Tg-IGF1R蛋白分子量为176.01 ku,等电点为6.04,包含4个Ⅲ型纤粘连蛋白结构域,1个半胱氨酸富集区和1个酪氨酸激酶结构域,属于亲水性跨膜蛋白质。氨基酸多重比对显示Tg-IGF1R蛋白序列与人、非洲爪蟾、斑马鱼的相似性分别为45.2%、45.6%和45.4%。利用q RT-PCR技术检测了Tg-IGF1R基因在泥蚶成体不同组织及幼体不同发育时期的表达情况,发现该基因具有广泛的组织表达性,在所检测的6个组织(血液、闭壳肌、斧足、鳃、内脏团和外套膜)中均有不同程度的表达,其中血液中的相对表达量最高,并与其余5个组织间存在显著性差异,推测Tg-IGF1R以信号分子的形式参与多种组织细胞的生命过程。Tg-IGF1R基因在泥蚶个体发育过程中的9个时期均有表达,且高表达主要集中在原肠胚期,其次是担轮幼虫期和稚贝期,说明Tg-IGF1R基因可能在早期发育阶段参与了某些器官的形成。

Cloning and expression analysis of IGF1R gene in Tegillarca granosa

On the basis of cDNA library of Tegillarca granosa, Tg-IGFIR cDNA was cloned by SMART RACE(Rapid Amplification of cDNA Ends) techniques. The full length cDNA of Tg-IGFIR was 5793 bp, and its complete ORF (Open Reading Frame) was 4638bp encoding 1546 amino acids. Homologous analysis on the amino acids showed that IGFIR of T. granosa shared more than 40% similarity with the most species of IGFIR, including Homo sapiens, Xenopus laevis, and Danio rerio. qRT-PCR results indicated that Tg-IGFIR gene was expressed in 6 tissues (blood, adductor muscle, foot, gill, visceral mass and mantle.), and the expression of blood showed significantly higher than other tissues, which implied that Tg-IGFIR plays various biological roles. The results of relative expression in 9 development stages revealed that the expression of Tg-IGFIR gene showed the highest expression in the gastrulae stage, and followed by trochophore and juvenile clams stages. Therefore, Tg-IGFIR may be involved in the formation of certain organs in early developmental stages.