奥利亚罗非鱼髓样分化因子(MyD88)的克隆及其在组织中的表达

Cloning and Expression of A Myeloid Differentiation Factor 88 Gene in Tilapia Oreochromis aureus

髓样分化因子（myeloid differentiation factor88，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1611bp，其中包括155bp的5’非编码区，589bp的3’非编码区和867bp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，其中表达最最高的是卵巢，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。

As an important adaptor protein in the intracellular signaling of Toll-like receptor superfamily, myeloid differentiation factor 88 （MyD88） plays a crucial role in the innate immune response. The full eDNA sequence of MyD88 （GenBank Acession number： JN032017） has been isolated by RACE-PCR method in Tilapia（ Oreochromis aureus）. The MyD88 gene is 1 611 bp, including a 155 bp 5＇-terminal untranslated region （UTR） , a 589 bp 3＇-UTR and an 867 bp open reading frame （ORF） , which encodes a polypeptide of 288 amino acids. The MyD88 protein contains the typical TLR and IL-1R-related （TIR） domain and death domain （DD）. Homology analysis revealed that the predicted amino acid sequence of Oreochromis aureus MyD88 shared the highest identity 85.8% to Siniperca chuats, 70% - 82% to other fishes, and 63% - 66% to mammals. The phylogenetic tree based on MyD88 proteins from 15 species showed that O. aureus shared the closest relationship with Siniperca chuatsi and Larimichthys crocea, all pertaining to Perciformes. Expression of MyD88 was determined by real-time RT-PCR （qRT-PCR） in different tissues. The results revealed that MyD88 transcripts were expressed in all tested tissues, being strongly expressed in the ovary, moderately expressed in intestine, spleen, liver, kidney, gill and blood, and weakly expressed in muscle and testis. This study will lay a foundation for further exploring the role of MyD88 in the TLR signaling in Tilapia.