斜带石斑鱼MyD88基因的克隆与表达

Cloning and Expression of MyD88 Gene in Orange-spotted Grouper Epinephelus coioides

本研究运用RACE-PCR技术获得斜带石斑鱼(Epinephelus coioides)髓样分化因子88(myeloid differentiation factor 88,MyD88)基因,并对该基因进行生物信息学和表达模式分析。研究结果表明1795bp的cDNA全长序列,包括ORF 870 bp、5'UTR 243 bp和3'UTR 682 bp,3'UTR存在1个多聚腺苷酸加尾信号(AATAAA)和两个mRNA不稳定基序(ATTTA)。SMART软件预测该蛋白N端和C端分别存在死亡结构域和TIR结构域(Toll/IL-1 receptor homology domain,TIR);与其它脊椎动物MyD88的序列同一性达57.1%～78.7%;用NJ法构建的系统进化树中,斜带石斑鱼MyD88和其它已报导的鱼类MyD88聚为一枝。qPCR检测结果显示MyD88基因mRNA主要表达于肝脏、脾脏、头肾和胸腺等组织。本研究为进一步探讨MyD88在斜带石斑鱼TLR信号传导中的作用奠定基础。

The full cDNA sequence of myeloid differentiation factor 88（MyD88） in the orange-spotted grouper Epinephelus coioides,designated as EcMyD88,has been isolated by using RACE-PCR method.The EcMyD88 consisted of 1 795 bp,including an 870 bp open reading frame（ORF）,a 243 bp 5＇ UTR and a 682 bp 3＇ UTR.The 3＇ UTR contained a polyadenylation-tailed signal（AATAAA） and 2 mRNA instable motifs（ATTTA）.The EcMyD88 protein comprised a death domain on N terminal and a TIR domain on C terminal by using SMART program.Putative amino acid sequence of EcMyD88 shared 57.1%～78.7% identity with its counterparts from other vertebrates.Phylogenetic tree was constructed by using neighbor-joining method which revealed that EcMyD88 was clustered with MyD88 from other teleost fish reported previously.The transcription of EcMyD88 was examined by real-time quantitative PCR,and its mRNA was mainly expressed in liver,spleen,thymus and head kidney.This study will lay a foundation for further exploring the role of MyD88 in the TLR signalling in Epinephelus coioides.