泥蚶(Tegillarca granosa)生长因子受体结合蛋白2(GRB2)基因的克隆与表达分析

CLONING AND EXPRESSION OF GRB2 GENE FROM THE BLOOD CLAM TEGILLARCA GRANOSA

通过已构建的泥蚶(Tegillarca granosa)转录组文库,利用SMART RACE技术扩增得到泥蚶Tg-GRB2基因的全长cDNA序列,并对其生物信息学、组织表达及发育阶段表达特征进行了分析。结果表明,泥蚶Tg-GRB2 cDNA全长1283bp,开放阅读框为708bp,编码236个氨基酸;蛋白结构预测显示,Tg-GRB2蛋白包含SH3-SH2-SH3三个功能域,SH2结构域由两端的α-螺旋和中间反向平行的β-折叠片构成,SH3结构域主要由β-折叠片组成,具备典型的结构特征;氨基酸序列比对发现,Tg-GRB2与脊椎动物的同源性为62.1%—63.8%,而与玻璃海鞘和日本血吸虫同源性较低。qRT-PCR检测结果显示:Tg-GRB2 mRNA在泥蚶血液、斧足、鳃、外套膜、闭壳肌、内脏团6个组织中都有表达,而在血液中的表达量极显著地高于其它组织;在泥蚶的不同发育阶段中,Tg-GRB2 mRNA在2—4细胞胚胎、原肠胚、担轮幼虫、D形幼虫中均有较高的表达量,而在D形幼虫中表达量最高,表明Tg-GRB2基因在泥蚶早期发育中发挥重要调节作用。

On the basis of 454 cDNA library of Tegillarca granosa, Tg-GRB2 cDNA was cloned by SMART RACE （Rapid Amplification of cDNA Ends） techniques and then the bioinformatics and expression profiles in different tissues and developmental stages were analyzed. The full length cDNA of Tg-GRB2 gene was 1275bp, containing a complete ORF （Open Reading Frame） encoding 236 amino acids. There were three functional domains of Tg-GRB2 protein （SH3- SH2-SH3）. The core structure of the SH2 domain contains an ant parallel ？-sheet and two ？-helices, and the structure of the SH3 domain was mainly composed by ？-sheets, which is similar to known GRB2 structure of mammalian. Homologous analysis showed that the amino acid sequence of Tg-GRB2 shared 62.1%—63.8% similarity with those of vertebrates, and relatively low homology with Ciona intestinalis and Schistosoma japonicum. The results of tissue-specific expression by real time PCR show that Tg-GRB2 expressed in all six tissues （blood, foot, gill, mantle, adductor muscle, and visceral mass）, and the expression of blood was significantly higher than those of other tissues. The results of relative expression in different development stages revealed that the expression of D larvae was higher than those of stages of 2—4 cells, gastrula, trochophore and juvenile clams. Therefore the Tg-GRB2 gene might play an important role in the formation of tissue and organs.