半滑舌鳎IRF-7基因的全长cDNA克隆及表达分析

Molecular cloning and tissue expression of IRF-7 gene of Cynoglossus semilaevis

中文关键词: 半滑舌鳎  干扰素调节因子7  Poly I:C  基因克隆  表达分析

英文关键词: Cynoglossus semilaevis  Interferon regulatory factor-7  Poly I:C  Gene cloning  Expression

中文摘要:

      本研究克隆了半滑舌鳎干扰素调节因子7 (CsIRF-7) cDNA的全长序列，并对该基因进行了结构特征分析，同时还完成了该基因在半滑舌鳎各组织中表达水平的定量分析。结果表明，CsIRF-7基因cDNA全长为1 957 bp，其中包含48 bp的5′-UTR、1 302 bp的ORF和607 bp的3′-UTR。CsIRF-7 cDNA编码一个由433个氨基酸残基组成的多肽。氨基酸序列比对结果表明，CsIRF-7多肽与脊椎动物IRF-7相似，存在高度保守的3个结构域：DNA结合域(DNA binding domain，DBD)、干扰素相关区(IRF associated domain，IAD)和丝氨酸富含区(Serine-rich domain，SRD)。全长氨基酸序列同源性分析发现，CsIRF-7与硬骨鱼类的IRF-7同源性较高，与4种硬骨鱼类的同一性和相似性分别介于48.5%～73.6%和65.4%～83.8%之间；其中与牙鲆的同一性和相似性最高，分别为73.6%和83.8%；而与哺乳类和鸟类IRF-7的同源性较低，其同一性和相似性分别仅为30.3%～32.2%和46.5%～52.5%。系统进化分析表明，CsIRF-7与硬骨鱼类的IRF-7聚为一类，并与其他脊椎动物的IRF-7和IRF-3共同组成了脊椎动物IRF-3亚家族。实时荧光定量PCR检测结果显示，CsIRF-7基因在半滑舌鳎的肝、肾、脾、血细胞、鳃、肠和心脏等7种检测的组织中均有表达，在肠中表达量最高，而在肝、鳃和血细胞中表达量相对较少。

英文摘要:

      The full-length cDNA sequence of interferon regulatory factor 7 (IRF-7) of tongue sole Cynoglossus semilaevis was cloned by rapid amplification cDNA end (RACE) method. The full-length cDNA of C. semilaevis IRF-7 (CsIRF-7) is 1957bp, containing a 48 bp 5′-untranslated region (5′-UTR), 1302 bp open reading frame (ORF), and a 607 bp 3′-UTR. The putative amino acid sequence of CsIRF-7 contains 433 amino acid residues. Through protein sequence alignment of IRF-7 in vertebrates,we found that the IRF-7 contains three completely conserved residues across all species,which are DNA-binding domain (DBD), IRF associated domain (IAD) and serine-rich domain (SRD). In contrast to IRF-7 of vertebrates other than fish, the DBD which is highly conserved has five tryptophan repeats, while CsIRF-7 and other fish IRF-7s have only four tryptophans with the second tryptophan residue missing. The deduced amino acid sequence of CsIRF-7 exhibited a high-level of homology with other fish, and shared the highest similarity (83.8%) and identity (73.6%) with the IRF-7 of Paralichthys olivaceus. The CsIRF-7 shared a low-level of homology with mammalia and aves. Phylogenetic analysis revealed that the C. semilaevis IRF-7 was clustered into IRF-7 subgroup with fish IRF-7 and formed the IRF-3 subfamily with IRF-3 group and other vertebrates IRF-7. Quantitative real-time method was used to detect the CsIRF-7 mRNA expression in different tissues. The results showed that CsIRF-7 was expressed in all tested tissues including liver, kidney, spleen, gill, intestine, blood cells, and heart. The high level expression of CsIRF 7 is detected in the intestine, heart, kidney, and spleen, while a lower-level expression in the liver, gill, and blood cells. These results will be helpful for further studies on functions of antiviral and antibacterial of CsIRF-7 gene.