【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Sparus aurata、大麻哈鱼Oncorhynchus keta、鲤鱼Cyprinus carpio和斑马鱼Danio rerio 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Oreochromis niloticus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。更多还原

【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 Sparus aurata,Oncorhynchus 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,Oncorhynchus 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.更多还原