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热带海洋学报 ›› 2009, Vol. 28 ›› Issue (6): 95-102.doi: 10.11978/j.issn.1009-5470.2009.06.095cstr: 32234.14.j.issn.1009-5470.2009.06.095

• 海洋生物学 • 上一篇    下一篇

斜带石斑鱼脂肪酸去饱和酶和延长酶基因全长cDNA序列的克隆与分析

李观贵, 梁旭方, 何珊, 郁颖, 陈晓艳, 黄柏炎, 程龙球   

  1. 暨南大学生命科学技术学院, 广东 广州 510632
  • 收稿日期:2007-12-26 修回日期:2008-07-25 出版日期:2009-12-30 发布日期:2009-12-12
  • 通讯作者: 梁旭方
  • 作者简介:李观贵(1983—)，男，广东茂名人，硕士，主要从事鱼类分子生物学研究。E-mail: liguangui@yahoo.com
  • 基金资助:

    国家科技部863项目(2007AA09Z437)；国家自然科学基金项目(30670367)；广东省科技计划项目(2005B20301005)；广东省自然科学基金项目(031886)

Molecular cloning and analysis of fatty acid desaturase and elongase genes in orange-spotted grouper (Epinephelus coioides)

LI Guan-gui, LIANG Xu-fang, HE Shan, YU Ying, CHEN Xiao-yan, HUANG Bo-yan, CHENG Long-qiu   

  1. College of Life Science and Technology, Jinan University, Shipai, Guangzhou 510632, China
  • Received:2007-12-26 Revised:2008-07-25 Online:2009-12-30 Published:2009-12-12
  • Contact: 梁旭方
  • About author:李观贵(1983—)，男，广东茂名人，硕士，主要从事鱼类分子生物学研究。E-mail: liguangui@yahoo.com
  • Supported by:

    国家科技部863项目(2007AA09Z437)；国家自然科学基金项目(30670367)；广东省科技计划项目(2005B20301005)；广东省自然科学基金项目(031886)

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被引次数

6 | 1

摘要：

利用逆转录聚合酶链式反应(reverse transcription polymerase chain reaction, RT-PCR)和cDNA末端快速扩增 (rapid-amplification of cDNA ends, RACE)方法获得斜带石斑鱼(Epinephelus coioides)肝脏中控制高不饱和脂肪酸合成的脂肪酸去饱和酶(fatty acid desaturase, FAD)和脂肪酸延长酶(fatty acid elongase, ELO)基因的全长cDNA序列。结果表明，FAD基因的全长cDNA序列长1979 bp，含1338 bp的开放阅读框（open reading frame, ORF），编码445个氨基酸。编码的蛋白序列含有FAD全部的特征结构区，包括3个组氨酸簇、2个跨膜区和1个细胞色素b5结构域，与金头鲷、大麻哈鱼、鲤鱼和斑马鱼FAD的氨基酸同源性为66.5%—90.8%。二级结构分析显示其以螺旋和β折叠为主，系统进化分析表明其与金头鲷和欧洲鲈鱼的亲缘关系最近。ELO基因的全长cDNA序列1228 bp，含有885 bp的开放阅读框，编码294个氨基酸。该蛋白序列含有单一的氧化还原中心组氨酸簇、内质网停留信号和多个跨膜区等ELO特征结构区，与金头鲷、大麻哈鱼、尼罗罗非鱼和斑马鱼ELO的氨基酸同源性为71.1%—85.7%。蛋白二级结构预测表明其含有丰富的螺旋和β折叠结构，系统树分析显示其与金头鲷亲缘关系最近。斜带石斑鱼FAD和ELO全长cDNA序列的获得，为今后进一步研究其高不饱和脂肪酸合成途径奠定基础。

关键词: 脂肪酸去饱和酶, 脂肪酸延长酶, 基因克隆, 斜带石斑鱼 (Epinephelus coioides)

Abstract:

Full-length cDNAs of fatty acid desaturase (FAD) and fatty acid elongase (ELO) genes, which were involved in the biosynthesis of highly unsaturated fatty acids (HUFA), were isolated from orange-spotted grouper (Epinephelus coioides) by RT-PCR (reverse transcription polymerase chain reaction) and RACE (rapid-amplification of cDNA ends) methods. The results revealed that the FAD cDNA was 1979 bp, including an ORF (open reading frame) of 1338 bp specified a protein of 445 amino acids. The deduced amino acid sequence shared 66.5%-90.8% identity with other fishes compared to Sparus aurata, Oncorhymchus keta, Cyprinus carpio and Danio rerio. Analysis online showed that its secondary structure was mainly composed helix and extended-beta, and phylogenetic tree revealed that it clustered closely with Sparus aurata and Dicentrarchus labrax. The protein sequence contained all the characteristics of microsomal FADs, including three histidine boxes, two transmembrane regions, and a cytochrome b5 domain. The 1228 bp cDNA of ELO included 885 bp ORF encoding a 294 amino acid peptide that contained a single histidine box redox centre motif, a canonical ER retention signal and multiple transmembrane regions similar with other ELOs. This protein sequence had 71.1%-85.7% identity with Sparus aurata, Oncorhymchus keta, Oreochromis niloticus and Danio rerio. Analysis online showed that its secondary structure was rich with helix and extended-beta, and phylogenetic tree revealed that it clustered closely with Sparus aurata. These results will help future study on the HUFA biosynthesis pathway in orange-spotted grouper.

Key words: fatty acid desaturase, fatty acid elongase, gene cloning, orange-spotted grouper (Epinephelus coioides)