泥鳅和大鳞副泥鳅细胞色素P450c17-Ⅰ(CYP17-Ⅰ)基因的克隆及组织表达分析

采用3'-和5'-RACE法克隆了泥鳅和大鳞副泥鳅CYP17-Ⅰ基因的cDNA全长序列,通过实时荧光定量PCR技术分析其表达情况。结果表明,泥鳅CYP17-Ⅰ基因cDNA全长1 706 bp,开放阅读框(open reading frame, ORF) 1 563 bp,编码520个氨基酸;大鳞副泥鳅CYP17-Ⅰ基因cDNA全长1 763 bp,ORF长1 545 bp,编码514个氨基酸。2种鳅CYP17-Ⅰ氨基酸序列都有1个信号肽、1个跨膜区、1个保守的蛋白结构域和3个功能保守区。相似度分析显示,2种鳅之间CYP17-Ⅰ相似度为99%,与其他鱼类的相似度也超过70%。系统进化分析显示,2种鳅之间关系最为接近,其系统发育关系基本符合传统的分类地位。qRT-PCR结果显示,CYP17-Ⅰ在2种鳅的肠、肌肉、心脏、胃、肝脏、精巢、卵巢、脾脏等8个组织均有表达,在精巢和卵巢中表达量相对较高。

Cloning and tissue expression analysis of the cytochrome P450c17-Ⅰ (CYP17-Ⅰ) gene from mud loach (Misgurnus anguillicaudatus) and large-scale loach (Paramisgurnus dabryanus)

In this study, full-length cDNA sequences of the cytochrome P450c17-Ⅰ (CYP17-Ⅰ) gene were cloned using homologous cloning and 3'- and 5'-rapid amplification of cDNA ends techniques from mud loach (Misgurnus anguillicaudatus) and large-scale loach (Paramisgurnus dabryanus). Their expression in various tissues were then explored using quantitative real-time PCR (qRT-PCR). In mud loach, the cloned sequence was 1 706 bp in length and contained a 1 563 bp open reading frame (ORF) encoding a protein of 520 amino acids. In the large-scale loach, the cloned sequence was 1 763 bp in length and contained a 1 545 bp ORF encoding a protein of 514 amino acids. Both CYP17-Ⅰ proteins had a predicted signal peptide, a transmembrane domain, a conserved protein domain and three conserved functional areas. Identity analysis showed that CYP17-Ⅰ amino acid sequences were 99% identical between the two loaches, and were more than 70% identical to the same proteins from other fish species. Phylogenetic analysis showed that the closest relationship of CYP17-Ⅰ amino acid sequences was between this two loaches, and the phylogenetic relationships among the CYP17-Ⅰ amino acid sequences were in accord with the traditional classification. CYP17-Ⅰ was expressed widely in the intestine, muscle, heart, stomach, liver, testis, ovary and spleen in the two species of loach, with the highest expression levels being observed in the the testis and ovary.