文蛤生长因子受体结合蛋白2(GRB2)基因克隆、时空表达及SNP位点筛查

Cloning,spatiotemporal expression and SNPs identification of GRB2 gene in hard clam Meretrix meretrix

为探索贝类GRB2基因的结构、功能特征,以及在贝类生长发育过程中的作用,实验通过已构建的文蛤转录组文库,利用SMART RACE技术扩增得到了文蛤GRB2基因的c DNA全长序列,对其生物信息学、组织及发育阶段表达特征进行了分析,并利用直接测序方法在外显子中筛选SNP位点。结果显示,GRB2基因c DNA全长1 791 bp,开放阅读框669 bp,编码223个氨基酸,蛋白由SH-SH2-SH3 3个结构域组成;氨基酸序列比对发现,文蛤G RB2与泥蚶的同源性最高,达到65.6%,与脊椎动物的同源性都在60%以上,说明GRB2基因在进化过程中比较保守。荧光实时定量PCR(qRT-PCR)结果表明,GRB2在文蛤6个组织和10个发育时期中均有表达,但不同组织间的表达量并没有显著差异;发育时期中的表达量呈现逐渐升高的趋势,在壳顶幼虫时期达到最高,之后表达量又有所降低。SNP位点的筛选结果表明,在G RB2基因的外显子区域共发现16个SNP位点。

To explore the molecular structure and biological function of G RB2 in Meretrix meretrix,G RB2 c DNA was cloned by SM ART RACE techniques,then the bioinformatics and expression profiles in different tissues and developmental stages were analyzed and SNPs were screened out in exon. The results indicated that the full length c DNA of G RB2 gene was 1 791 bp,containing a complete 669 bp ORF encoding 223 amino acids. There were three functional domains of G RB2 protein（ SH3-SH2-SH3）. Comparisons of animal acid sequence,the G RB2 of M. meretrix has highly homologous with Tegillarca granosa and shares 65. 6%similarity. It shares more than 60% similarity with vertebrates and proves that G RB2 was highly conservative. The result of qRT-PCR showed that G RB2 expressed in all six tissues and ten developmental stages,but the expression of tissues did not have significantly difference. The relative expression in different stages revealed that the expression of G RB2 gradually increased with the process of the development,and showed the highest in umbo larvae stage. 16 SNPs in the exon of G RB2 were identified.