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

Cloning and expression analysis of IGF1R gene in Tegillarca granosa

  • 摘要: 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)cDNA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qRT-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。

     

    Abstract: In order to explore the function of IGF1R during the process of the growth and development in Tegillarca granosa, Tg-IGF1R cDNA was cloned by SMART RACE (Rapid Amplification of cDNA Ends) techniques on the basis of cDNA library of T.granosa. The full length cDNA of Tg-IGF1R was 5793 bp, and its complete ORF (Open Reading Frame) was 4638 bp encoding 1546 amino acids. Bioinformatics analysis showed that the sequence of Tg-IGF1R contains a 5'-untranslated region (UTR) of 810 bp, and a 3'-UTR of 345 bp. The predicted molecular mass of the deduced amino acid of Tg-IGF1R was 176.01 ku, and the theoretical isoelectric point was 6.04. Tg-IGF1R protein consists of four fibronectin type Ⅲ(FN3) domains, one furin like cysteine-rich (FU) domain and Tyrosine kinase (TyrKc) domain, belonging to transmembrane and hydrophilic protein. Homologous analysis on the amino acids showed that IGF1R of T. granosa shared more than 40% similarity with the most species of IGF1R, including Homo sapiens, Xenopus laevis, and Danio rerio. qRT-PCR (Real-time quantitative PCR) results indicated that Tg-IGF1R gene was expressed in 6 tissues (blood, adductor muscle, foot, gill, visceral mass and mantle.), and the expression of blood was significantly higher than other tissues, which implied that Tg-IGF1R plays various biological roles. The results of relative expression in 9 development stages revealed that the expression of Tg-IGF1R gene showed the highest expression in the gastrulae stage, and followed by trochophore and juvenile clams stages. Therefore, Tg-IGF1R may be involved in the formation of certain organs in early developmental stages.

     

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