半滑舌鳎髓样分化因子的克隆和表达分析

Cloning,characterization and expression of a myeloid differentiation factor 88(MyD88)gene in half smooth tongue sole,Cynoglossus semilaevis

髓样分化因子(Myeloid differentiation factor88,MyD88)是Toll/IL-lR家族成员,是TLR信号通路中的一个关键接头分子,在天然免疫系统中具有重要作用。迄今,在重要海水养殖鱼类半滑舌鳎中尚未见有关MyD88的研究报道。本研究克隆了半滑舌鳎(Cynoglossus semilaevis)MyD88基因,并对其表达模式进行了初步研究。半滑舌鳎MyD88基因的cDNA全长为1612bp,其中包括132bp的5′非翻译区(UTR),590bp的3′UTR和编码285个氨基酸残基的858bp的开放阅读框(ORF)。MyD88蛋白在N端具有死亡结构域(Death Domain,DD),C端具有TIR结构域(Toll/IL-1Receptor Domain,TIR),MyD88基因组全长为2923bp,包括5个外显子和4个内含子。系统进化树分析表明,半滑舌鳎MyD88和牙鲆聚在一起,具有最近的亲缘关系。实时定量PCR结果显示,MyD88基因几乎在所有组织中都有表达,在血液、脾脏、头肾、肝脏等免疫组织和精巢、卵巢生殖腺中具有较高水平的表达。利用LPS(lipopolysaccharide)、PGN(peptidoglycan)和poly I∶C作为免疫刺激源感染半滑舌鳎外周血淋巴细胞的结果显示,三者均可诱导MyD88基因的表达上调。鳗弧菌(Vibrio angaillarum)感染实验表明,注射鳗弧菌96h后MyD88基因在脾脏中表达量升高了近180倍、在肝脏中的表达量升高了60多倍;在注射鳗弧菌48h后,头肾中表达量升高了近5倍。上述实验暗示,MyD88基因在半滑舌鳎天然免疫防御系统中具有重要作用。

As an important adaptor protein in the intracellular signaling of interleukin-1 receptor/toll-like receptor （IL-1R/TLR） superfamily,myeloid differentiation factor 88（ MyD88） plays a crucial role in the innate immune response. So far,no report about MyD88 in half smooth tongue sole（ Cynoglossus semilaevis）,one of the most promising and commercially important species for marine fish culture in China,is available. In this paper,the cloning,characterization and expression pattern of MyD88 in half smooth tongue sole was described. The full length MyD88 cDNA of half smooth tongue sole is 1 612 bp including a 132 bp 5′ untranslated region（ UTR）,a 590 bp 3′ UTR and a 858 bp open reading frame（ ORF） encoding 285 amino acids. MyD88 contains a typical death domain at the N-terminal and a conservative Toll/IL-1R（ TIR） domain at the C-terminal. The length of genomic DNA of MyD88 in this species is 2 923 bp with 5 exons and 4 introns. The phylogenetic tree based on 11 species MyD88 proteins showed that half smooth tongue sole MyD88 shared the closetest relationship with Japanese flounder（ Paralichthys olivaceus）. The next quantitative real-time reverse transcription PCR analysis revealed MyD88 gene was expressed in all tested tissues with higher expression in immune tissues and reproduction tissues including blood,liver,spleen, kidney,testis and ovary. Then,the stimulation with LPS（ lipopolysaccharide）,GPN（ peptidoglycan） and poly I：C showed that these stimulators could induce up-regulation of MyD88 expression in PBL of half smooth tongue sole. Finally,the pathogen infection experiment with Gram negative bacteria Vibrio anguillarum,indicated that MyD88 expression was significantly increased in the spleen by nealy 180 times and in the liver by more than 60 times after 96 th hour and in the headkidney with near 5 times at 48 th hour after challenge,respectively. These results suggest that MyD88 play an important role in the innate immune system in half smooth tongue sole.