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

doi:10.3969/j.issn.1004-1524.2016.11.08

浙江农业学报 ›› 2016, Vol. 28 ›› Issue (11): 1853-1861.DOI: 10.3969/j.issn.1004-1524.2016.11.08

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

刘士力^{1, 2}, 张爱菊¹, 练青平¹, 吴卫君^{3, 4, *}, 贾永义¹, 蒋文枰¹, 李飞¹

1.浙江省淡水水产研究所农业部淡水渔业健康养殖重点实验室/浙江省淡水水产遗传育种重点实验室,浙江湖州313001;
2.上海海洋大学水产与生命学院,上海201306;
3.庆元县水利局水产技术推广站,浙江庆元323800;
4.庆元县水电学会水产分会,浙江庆元323800

收稿日期:2016-03-14 出版日期:2016-11-15 发布日期:2016-11-16
通讯作者: 吴卫君,E-mail:zjwuweijun@163.com
作者简介:刘士力(1985—),男,湖北洪湖人,博士研究生,主要从事水生动物遗传育种研究。E-mail:liushili1212@126.com
基金资助:
浙江省淡水水产遗传育种重点实验室建设(2014F10037); 浙江省重大科技专项(2013C02009); 浙江省设施水产养殖科技创新团队项目(2011R50029); 浙江省公益技术研究项目(2010C32033,2012C22059); 庆元县科技项目(QKF2015-22-8)

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

LIU Shi-li^{1, 2}, ZHANG Ai-ju¹, LIAN Qing-ping¹, WU Wei-jun^{3, 4, *}, JIA Yong-yi¹, JIANG Wen-ping¹, LI Fei¹

1. Zhejiang Institute of Freshwater Fisheries, Agriculture Ministry Key Laboratory of Healthy Freshwater Aquaculture / Key Laboratory of Freshwater Aquatic Animal Genetic and Breeding of Zhejiang Province, Huzhou 313001, China;
2. College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China;
3. Fisheries Technology Extension Station, Water Resources Bureau of Qingyuan County, Qingyuan 323800, China;
4. Fisheries Branch, Hydropower Institute of Qingyuan County, Qingyuan 323800, China

Received:2016-03-14 Online:2016-11-15 Published:2016-11-16

摘要/Abstract

摘要： 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。

关键词: 细胞色素, 泥鳅, 大鳞副泥鳅, CYP17-Ⅰ

Abstract: 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.

Key words: cytochrome, Misgurnus anguillicaudatus, Paramisgurnus dabryanus, CYP17-Ⅰ

中图分类号:

S917.4

刘士力, 张爱菊, 练青平, 吴卫君, 贾永义, 蒋文枰, 李飞. 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[J]. 浙江农业学报, 2016, 28(11): 1853-1861.

LIU Shi-li, ZHANG Ai-ju, LIAN Qing-ping, WU Wei-jun, JIA Yong-yi, JIANG Wen-ping, LI Fei. Cloning and tissue expression analysis of the cytochrome P450c17-Ⅰ (CYP17-Ⅰ) gene from mud loach (Misgurnus anguillicaudatus) and large-scale loach (Paramisgurnus dabryanus)[J]. , 2016, 28(11): 1853-1861.

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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

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

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