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髓样分化因子(myeloid differentiation factor 88,MyD88)是TLR(toll-like receptor)信号通路的关键接头蛋白,在先天性免疫中具有重要作用。通过RACE-RCR技术克隆了奥利亚罗非鱼(Oreochromis aureus)MyD88基因cDNA全长序列(GenBank登录号:JN032017)。序列分析表明,奥利亚罗非鱼MyD88基因全长为1 611 bp,其中包括155 bp的5’非编码区,589 bp的3’非编码区和867 bp的编码区,编码288个氨基酸残基。MyD88蛋白N端具有死亡结构域,C端具有TIR结构域。同源性分析表明,奥利亚罗非鱼MyD88氨基酸序列与鳜鱼(Siniperca chuats)相似性最高,为85.8%,与其他鱼类相似性为70%~82%,与哺乳动物相似性为63%~66%;系统进化树分析表明,奥利亚罗非鱼MyD88与同属鲈形目的鳜鱼、大黄鱼(Larimichthys crocea)聚在一起。采用实时定量PCR方法检测MyD88在奥利亚罗非鱼各组织中的表达情况。结果显示,MyD88在所有被测组织中都有表达,其中表达量最高的是卵巢,其次在小肠、脾、肝、肾、鳃和血液中有较高的表达量,肌肉、精巢组织中表达量最低。本研究可为进一步探讨MyD88在奥利亚罗非鱼TLR信号通路中的作用奠定一定的基础。
Myeloid differentiation factor 88 (MyD88) is a key adapter protein in the toll-like receptor (TLR) signaling pathway and plays an important role in innate immunity. The full-length cDNA of MyD88 gene from Oreochromis aureus was cloned by RACE-RCR (GenBank accession number: JN032017). Sequence analysis showed that the MyD88 gene of O. tilapia was 1 611 bp in length, including 155 bp 5 ’non-coding region, 589 bp 3’ non-coding region and 867 bp coding region, encoding 288 amino acid residues base. The MyD88 protein has a death domain at the N-terminus and a TIR domain at the C-terminus. Homology analysis showed that the amino acid sequence of MyD88 of O. tilapia shared the highest similarity with Siniperca chuats (85.8%), similar to other fishes (70% -82%) and similar to mammals % ~ 66%. Phylogenetic tree analysis showed that MyD88 was associated with Larimichthys crocea, the same species of perciformes. Real-time quantitative PCR was used to detect the expression of MyD88 in tissues of Oreochromis aurea. The results showed that MyD88 was expressed in all the tested tissues. Among them, the expression of MyD88 was the highest in the ovary, followed by the expression in the intestine, spleen, liver, kidney, gill and blood, lowest. This study may provide a basis for further exploring the role of MyD88 in the TLR signaling pathway in O. aurea.