四种不同生境鱼类脂肪酸去饱和酶和延长酶基因cDNA全序列的克隆与分子进化分析
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摘要
利用RT-PCR和RACE方法克隆得到草鱼(Ctenopharyngodon idellus)、鳜鱼(Siniperca chuatsi)/日本鳗鲡(Anguilla japonica)和斜带石斑鱼(Epinephelus coioides)肝脏中控制高不饱和脂肪酸(HUFA)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cDNA全序列。FAD基因编码444至445个氨基酸,与其他鱼类序列有较高同源性(至少67%),主要的序列差异集中在N端和C端的多肽序列上。推测的FAD氨基酸序列含有脊椎动物Δ6/Δ5 FAD全部的特征结构区,包括保守的多个结构域：3个组氨酸簇,2个跨膜区域和1个含亚铁血红素结合基序的类似细胞色素b5结构域。ELO基因编码291至294个氨基酸,与其他鱼类序列有较高同源性(至少65%),主要的序列差异集中在C端的多肽序列上。推测的ELO氨基酸序列包括脊椎动物ELOVL5的所有特征结构功能区：单一的氧化还原中心组氨酸簇、1个内质网停留信号和4个ELO共有的保守区域等。使用4种不同的方法(NJ、MP、ML、BI)构建系统进化树结果显示,基于FAD和ELO的一致树类似,大致可以分为4支：食草性和杂食性淡水鱼聚为一支;溯河洄游型鱼为一支；降河洄游鱼单独为一支；海水鱼和食肉性淡水鱼聚为一支。多个鱼类FAD和ELO基因全长cDNA序列的获得为进一步研究鱼类HUFA合成能力及调控机理奠定基础。
Full-length cDNAs of fatty acid desaturase (FAD) and fatty acid elongase (ELO), which were involved in the biosynthesis of highly unsaturated fatty acids (HUFA), were isolated from grass carp(Ctenopharyngodon idellus), mandarin fish (Siniperca chuatsi), Japanese eel (Anguilla japonica), and orange-spotted grouper (Epinephelus coioides), based on RT-PCR and RACE methods. The open reading frames of four FAD genes encode peptides of 444 and 445 amino acids, along with those from other fish species, are similar and shared at least 67% sequence identity with each other, with most of the sequence variation residing in the N-and C-terminal region of the polypeptide. The deduced amino acid sequences is highly conserved that contains all the characteristics of vertebrateΔ6/Δ5 FAD, including three histidine boxes, two membrane-spaning domains, and one cytochrome b5-like domain with the typical heme-binding motif. The ORFs of four ELO genes encode peptides of 291 and 294 amino acids, along with those from other fish species, are similar and shared at least 65% sequence identity with each other, with most of the sequence variation residing in the C-terminal region of the polypeptide. The deduced amino acid sequences contain all the characteristics of vertebrate ELOVL5, including single histidine box redox centre motif, one canonical ER retention signal and multiple transmembrane regions. The phylogenetic trees based on FAD/ELO ORFs, using four different methods (NJ, MP, ML, BI) show that the consensus trees are similar. The phylogenetic sequence analyses grouped the teleost fish into four distinct clusters with the mainly herbivorous and omnivorous freshwater fish, anadromous fish, catadromous fish, seawater fish and carnivorous freshwater fish. These results will help the following study of the HUFA biosynthesis pathway in teleost fish.
Full-length cDNAs of fatty acid desaturase (FAD) and fatty acid elongase (ELO), which were involved in the biosynthesis of highly unsaturated fatty acids (HUFA), were isolated from grass carp(Ctenopharyngodon idellus), mandarin fish (Siniperca chuatsi), Japanese eel (Anguilla japonica), and orange-spotted grouper (Epinephelus coioides), based on RT-PCR and RACE methods. The open reading frames of four FAD genes encode peptides of 444 and 445 amino acids, along with those from other fish species, are similar and shared at least 67% sequence identity with each other, with most of the sequence variation residing in the N-and C-terminal region of the polypeptide. The deduced amino acid sequences is highly conserved that contains all the characteristics of vertebrateΔ6/Δ5 FAD, including three histidine boxes, two membrane-spaning domains, and one cytochrome b5-like domain with the typical heme-binding motif. The ORFs of four ELO genes encode peptides of 291 and 294 amino acids, along with those from other fish species, are similar and shared at least 65% sequence identity with each other, with most of the sequence variation residing in the C-terminal region of the polypeptide. The deduced amino acid sequences contain all the characteristics of vertebrate ELOVL5, including single histidine box redox centre motif, one canonical ER retention signal and multiple transmembrane regions. The phylogenetic trees based on FAD/ELO ORFs, using four different methods (NJ, MP, ML, BI) show that the consensus trees are similar. The phylogenetic sequence analyses grouped the teleost fish into four distinct clusters with the mainly herbivorous and omnivorous freshwater fish, anadromous fish, catadromous fish, seawater fish and carnivorous freshwater fish. These results will help the following study of the HUFA biosynthesis pathway in teleost fish.
引文
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