Department of Fisheries and Wildlife Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA.
Fish Shellfish Immunol. 2011 Jan;30(1):361-71. doi: 10.1016/j.fsi.2010.11.012. Epub 2010 Nov 16.
Induction of innate immune pathways is critical for early host defense, but there is limited understanding of how teleost fishes recognize pathogen molecules and activate these pathways. In mammals, cells of the innate immune system detect pathogenic molecular structures using pattern recognition receptors (PRRs). TLR9 functions as a PRR that recognizes CpG motifs in bacterial and viral DNA and requires adaptor molecules MyD88 and TRAF6 for signal transduction. Here we report full-length cDNA isolation, structural characterization and tissue mRNA expression analysis of the common carp (cc) TLR9, MyD88 and TRAF6 gene orthologs. The ccTLR9 open-reading frame (ORF) is predicted to encode a 1064-amino acid (aa) protein. We found that MyD88 and TRAF6 genes are duplicated in common carp. This is the first report of TRAF6 duplication in a vertebrate genome and stronger evidence in support of MyD88 duplication is provided. The ccMyD88a and b ORFs are predicted to encode 288-aa and 284-aa peptides, respectively. They share 91% aa sequence identity between paralogs. The ccTRAF6a and b ORFs are both predicted to encode 543-aa peptides sharing 95% aa sequence identity between paralogs. The ccTLR9 gene is contained in a single large exon. The ccMyD88a and ccMyD88b coding sequences span five exons. The TRAF6b gene spans six exons. PCR amplification to obtain the entire coding sequence of ccTRAF6a gene was not successful. The 2104-bp fragment amplified covers the 3' end of the gene and it contains a partial sequence of one exon and three complete exons. The predicated protein domains of the ccTLR9, ccMyD88 and ccTRAF6 are conserved and resemble orthologs from other vertebrates. Real-time quantitative PCR assays of the ccTLR9, MyD88a and b, and TRAF6a and b gene transcripts in healthy common carp indicated that mRNA expression varied between tissues. Differential expression of duplicate copies were found for ccMyD88 and ccTRAF6 in white and red muscle tissues, suggesting that paralogs may have evolved and attained a new function. The genomic information we describe in this paper provides evidence of sequence and structural conservation of immune response genes in common carp.
先天免疫途径的诱导对于早期宿主防御至关重要，但人们对硬骨鱼类如何识别病原体分子并激活这些途径知之甚少。在哺乳动物中，先天免疫系统的细胞使用模式识别受体 (PRR) 来检测致病性分子结构。TLR9 作为一种 PRR，可识别细菌和病毒 DNA 中的 CpG 基序，并需要衔接分子 MyD88 和 TRAF6 进行信号转导。在这里，我们报告了鲤鱼 (cc) TLR9、MyD88 和 TRAF6 基因直系同源物的全长 cDNA 分离、结构特征和组织 mRNA 表达分析。ccTLR9 开放阅读框 (ORF) 预测编码 1064 个氨基酸 (aa) 的蛋白质。我们发现 MyD88 和 TRAF6 基因在鲤鱼中重复。这是脊椎动物基因组中 TRAF6 重复的首次报道，并提供了更强的证据支持 MyD88 重复。ccMyD88a 和 b ORFs 分别预测编码 288-aa 和 284-aa 肽。它们在直系同源物之间具有 91%的 aa 序列同一性。ccTRAF6a 和 b ORFs 均预测编码 543-aa 肽，在直系同源物之间具有 95%的 aa 序列同一性。ccTLR9 基因包含在单个大外显子中。ccMyD88a 和 ccMyD88b 编码序列跨越五个外显子。TRAF6b 基因跨越六个外显子。PCR 扩增未能成功获得 ccTRAF6a 基因的全长编码序列。扩增获得的 2104-bp 片段覆盖基因的 3' 端，包含一个外显子的部分序列和三个完整外显子。ccTLR9、ccMyD88 和 ccTRAF6 的预测蛋白结构域保守，与其他脊椎动物的同源物相似。健康鲤鱼中 ccTLR9、MyD88a 和 b 以及 TRAF6a 和 b 基因转录本的实时定量 PCR 检测表明，mRNA 表达在组织之间存在差异。在白色和红色肌肉组织中发现了 ccMyD88 和 ccTRAF6 的重复拷贝的差异表达，表明了这些基因可能已经进化并获得了新的功能。我们在本文中描述的基因组信息提供了鲤鱼中免疫反应基因序列和结构保守性的证据。
