Tumor necrosis factor receptor-associated factor 6 (TRAF6) and transforming growth factor-β-activated kinase 1 (TAK1) are important adaptor molecules in Toll-like receptor (TLR) signaling pathways. To better understand the biological role of these two genes in immune response, we cloned TRAF6 and TAK1 cDNA sequences from half-smooth tongue sole (Cynoglossus semilaevis) using homologous cloning methods and rapid amplification of cDNA ends. We also detected the expression patterns of these two genes in different tissues at different developmental stages. The full-length cDNA sequence of TRAF6 was 1956 bp, including a 1731 bp open reading frame (ORF) that encoded a putative 576 amino acid protein. TRAF6 contained one RING domain, two zinc fingers, one coiled-coil region, and one MATH domain; this structure is highly similar to that of TRAF6 in other species. The conserved motifs of TRAF6 likely indicate that its functions are similar to those of other mammal TRAF6s. The full-length TAK1 cDNA sequence was 2519 bp, including a 1731 bp ORF that encoded a putative 576 amino acid protein. TAK1 contains a conserved serine/threonine protein kinase catalytic domain and a coiled-coil region. The highly conserved domains indicate that all TAK1s have a similar function. Phylogenetic trees showed that both TRAF6 and TAK1 in C. semilaevis were evolu-tionarily closest to those in Paralichthys olivaceus. In addition, the expression patterns of these two genes were exam-ined in different tissues and developmental stages. TRAF6 was expressed in all tested tissues, and the highest expression was in the gills followed by the intestines. TAK1 was highly expressed in the heart and kidney. The high levels of TRAF6 and TAK1 in the gills and kidneys were consistent with the essential role of the two genes in the TLR/Toll-like receptor signaling pathway, which is pivotal in both innate and adaptive immune responses. TRAF6 and TAK1 were expressed throughout developmental stages, including unfertilized eggs, indicating maternal inheritance of TRAF6 and TAK1. The expression of maternal mRNA throughout development demonstrates the potential role of TRAF6 and TAK1 in early immune defense and developmental regulation of C. semilaevis. These results indicate that TRAF6 and TAK1 may play crucial roles in immune responses and might be involved in half-smooth tongue sole development. This study provides a theoretical basis for understanding the roles of these two genes in C. semilaevis immune response.%本研究通过同源克隆和 RACE 技术获得了半滑舌鳎(Cynoglossus semilaevis)肿瘤坏死因子受体相关因子6(tumor necrosis factor receptor-associated factor 6, TRAF6)和转化生长因子β激活激酶1(transforming growth fac-tor-β-activated kinase 1, TAK1)的cDNA全长,并分析了其在不同组织和早期胚胎发育时期的表达情况。结果表明, TRAF6 cDNA全长1956 bp,开放阅读框(ORF)为1731 bp,编码576个氨基酸。二级结构预测显示TRAF6具有保守的蛋白结构域： N端的RING结构,两个锌指结构以及C端的环–环(coiled-coil)α螺旋结构和高度保守的MATH同源结构。TAK1 cDNA全长2519 bp, ORF 为1731 bp,编码576个氨基酸。TAK1的蛋白结构域包括丝氨酸/苏氨酸蛋白激酶激活结构域和C端的环–环(coiled-coil)α螺旋结构域。系统进化树分析表明,半滑舌鳎TRAF6和TAK1分别与牙鲆(Paralichthys olivaceus)TRAF6和TAK1聚为一支,亲缘关系最近。荧光定量PCR结果显示, TRAF6和TAK1在所检测的8种组织中都有表达, TRAF6在鳃中的表达最高,肠中也有较高的表达； TAK1在心脏中的表达量最高,其次是肾。TRAF6和TAK1在鳃、肾等免疫器官中的高表达,与其在Toll样受体信号通路中的重要作用是一致的。对TRAF6和TAK1在早期胚胎发育时期的表达情况进行检测,结果显示,在未受精卵中可检测到TRAF6和TAK1,提示了这两种免疫分子的母源性 mRNA 遗传的可能性。免疫分子母源性 mRNA 可能参与发育过程,也可能参与构建免疫体系,以保护胚胎或仔鱼免受病原体的侵袭。
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