马氏珠母贝Ago2基因克隆与表达分析

Cloning and Expression Analysis of Ago2 Gene in Pinctada fucata martensii

为探讨由Argonaute 2(Ago2)介导的LncRNA-miRNA调控系统在贝类生物矿化(Biomineralization)的调控机制,本研究利用RACE技术克隆获得马氏珠母贝(Pinctada fucata martensii)Ago2(PmAgo2)基因cDNA全长序列并检测其表达。结果表明,PmAgo2全长3465 bp,开放阅读框(open reading frame,ORF)2679 bp,编码892个氨基酸残基,5’UTR为113 bp,3’UTR为673 bp;预测其分子量为101.06139 kD,理论等电点为9.48;多序列比对的结果表明,PmAgo2与脊椎动物和其他无脊椎的Ago2具有较高的保守性,与美洲牡蛎(Crassostrea virginica)的相似度高达88%;qRT-PCR结果显示,PmAgo2在马氏珠母贝各个组织均有表达,外套膜边缘膜区的表达量最高。利用脊椎动物Ago2抗体,通过Western blotting检测其在外套膜组织中的表达,发现Ago2蛋白的分子量在100 kD左右,与理论的分子量相一致。本研究结果为后期深入研究由Ago2介导的LncRNA-miRNA非编码RNA调控系统奠定基础。

To explore the regulatory mechanism of LncRNA-microRNA regulatory system mediated by Argonaute2(Ago2)in shellfish biomineralization.In this study,the full-length cDNA sequence of the Ago2(PmAgo2)gene of Pinctada fucata martensii was cloned by using the RACE technique and its expression was detected.The results showed that the full length of PmAgo2 gene cDNA was 3465 bp,containing 2679 bp length of the open reading frame(ORF)which encoded 892 amino acid residues.The 5’UTR was 113 bp and the 3’UTR was 673 bp.The predicted molecular weight was 101.06139 kD and the theoretical isoelectric point was 9.48.The results of multiple sequence alignment indicated that PmAgo2 is highly conserved with vertebrates and other invertebrates,and the similar degree with Crassostrea virginica was as high as 88%.The results of qRT-PCR showed that PmAgo2 was expressed in each tissue of Pinctada fucata martensii,with its expression highest in the marginal zone.The study used the Ago2 antibody of vertebrates to test its expression in mantle tissue by Western blotting and found that the molecular weight of Ago2 protein was about 100 kD,which was consistent with the molecular weight of the theory.The results of this study lay a foundation for further study on the regulation system of non-coding RNA of lncRNA-miRNA mediated by Ago2.