泥鳅elovl2和elovl5基因克隆、表达及其延长活性分析

Cloning,expression analysis and functional study of loach(Misgurnus anguaudatus)elovl2 and elovl5 genes and their effects on the synthesis of long-chain polyunsaturated fatty acids(LC-PUFA)

为探究泥鳅(Misgurnus anguillicaudatus)脂肪酸延长酶2和延长酶5基因(elovl2,elovl5)在长链多不饱和脂肪酸(LC-PUFA)合成中的作用,克隆获得泥鳅elovl2和elovl5基因cDNA全长序列,分析其在泥鳅不同组织中的表达规律;通过酵母体外表达验证其延长活性;并投喂以鱼油(FO)、亚麻芥油(CO)和大豆油(SO)为脂肪源的饲料8周,分析不同脂肪源对泥鳅elovl2和elovl5表达的影响。结果显示,泥鳅elovl2和elovl5的开放阅读框(ORF)分别为888 bp和876 bp,编码295和291个氨基酸。系统进化树分析表明泥鳅ELOVL2、ELOVL5和斑马鱼的同源性最近。序列比对分析ELOVL2和ELOVL5保守性较高,都具有长链脂肪酸延伸酶的特殊结构。泥鳅elovl2和elovl5均在肝组织中表达量最高。酵母功能验证结果表明泥鳅ELOVL2主要延长C20-22 PUFA,而ELOVL5主要延长C18-20 PUFA,对C22 PUFA没有延长作用。不同饲料脂肪源投喂试验结果显示,CO组泥鳅肝脏和肌肉n-3 PUFA含量显著上升,肌肉20∶5n-3含量显著升高,但肝脏和肌肉22∶6n-3含量没有显著变化。CO组泥鳅肝组织中elovl2、elovl5、scd和srebp-1c的表达量也显著上升。综上所述,泥鳅ELOVL2和ELOVL5在LC-PUFA合成中具有重要作用,饲料中CO添加可促进泥鳅n-3 PUFA的积累。

To investigate roles of the long-chain fatty acid elongase 2 and 5(elovl2,elovl5)genes played in biosynthesis of long-chain polyunsaturated fatty acids(LC-PUFA)in loach(Misgurnus anguaudatus),full-length cDNA sequences of the loach elovl2 and elovl5 were cloned and their expression patterns in different tissues were analyzed in this study.Functional characterizations of the loach elovl2 and elovl5 in synthesis of fatty acids were studied by heterologous expression in Saccharomyces cerevisiae in vitro.Loach were fed with three experimental diets containing fish oil(FO),camelina oil(CO)and soybean oil(SO),respectively,for 8 weeks to evaluate the effects of different dietary lipid sources on expressions of the loach elovl2 and elovl5.The results showed that the open reading frame(ORF)of the loach elovl2 and elovl5 was 888 bp and 876 bp,encoding 295 and 291 amino acids,respectively.Phylogenetic analysis showed that they clustered closely with the zebrafish elovl2 and elovl5 and sequence comparison showed that they were highly conserved in vertebrates,containing the special structure of long-chain fatty acid elongases.The expression levels of elovl2 and elovl5 were highest in the liver.ELOVL2 elongated C20-22 PUFA,while ELOVL5 was able to elongate C18-20 PUFA,having no effect on C22 PUFA.The content of n-3 PUFA in the liver and muscle and 20∶5n-3 in the muscle increased significantly,while 22∶6n-3 in the liver and muscle did not increase significantly in the CO group.In addition,expressions of the loach elovl2,elovl5,scd and srebp-1c in the liver were significantly increased.In conclusion,elovl2 and elovl5 played an important role in regulating the synthesis of LC-PUFA and CO used in diet could promote the accumulation of n-3 PUFA in loach.