鲤鱼2种脂酰辅酶A脱氢酶基因cDNA克隆、表达及活性研究

Cloning,Expression and Activity Analysis of Two Kinds ACADs in Cyprinus carpio

为探究脂酰辅酶A脱氢酶对脂肪酸β氧化的调控机制,使用RT-PCR在鲤鱼肝脏克隆了脂酰辅酶A脱氢酶家族中的MCAD和LCAD全长c DNA序列,阅读框分别为1 275,1 326 bp,编码424,441个氨基酸,分析表明他们均具备ACADs家族的特征序列:FAD结合位点、底物结合位点、催化位点等,与斑马鱼MCAD和LCAD的一致性分别为93.16%和94.56%。实时定量RT-PCR结果表明,MCAD和LCAD主要在肝脏和心脏表达;不同脂肪源对MCAD的表达没有明显影响,但鱼油对LCAD的表达有明显的刺激作用。此外,构建了原核表达载体MCADs-p EASY(E2)和LCADs-pEASY(E2),经IPTG诱导获得了原核表达蛋白,分子量分别为43.0,43.6 kDa。经检测这2个蛋白在370,450 nm处有明显的吸收峰,表明他们能自然结合FAD辅基。吩嗪硫酸甲酯反应法结果表明:MCAD和LCAD酶活性最适温度均为28℃,酶活分别为9.12,10.29 U/g。结果表明,与哺乳动物类似,鲤鱼MCAD和LCAD在肝脏和心脏表达量高;高不饱和脂肪酸对LCAD的表达有促进作用。

To investigate the regulatory mechanism of fatty acids fatty Acyl coenzyme A （Acyl-CoA dehydrogenase,ACADs） on fatty acids β-oxidation in mitochondrial,this experiment cloned two genes,MCAD and LCAD,of ACADs family from the liver tissue of Cyprinus carpio using classic RT-PCR.The open reading frames of MCAD and LCAD were 1 275,1 326 bp in length respectively,encoding 424 and 441 amino acids with characteristics of ACADs family structure （such as FAO binding sites,the substrate binding site and catalytic site）,sharing high homology with zebrafish,93.16% and 94.56% respectively.The results of RT-PCR showed that MCAD and LCAD mainly expressed in the liver and heart. No significant influence was observed of different sources of fat on the expression of MCAD,while fish oil could obviously stimulate the expression of LCAD. After IPTG induction,both the two prokaryotic expression vectors,MCADs-pEASY（E2）,and LCADs-pEASY（E2）,expressed fusion proteins of 43.0,43.6 kDa in molecular weight,with obvious absorption peak at 370,450 nm,which was proved to be able to naturally bind to FAO cofactors. Phenazine methyl sulfate reaction method was applied to determine the enzyme activity of Prokaryotic expression protein,and the results indicated that the optimum temperature for MCAD and LCAD enzymes were 28℃,and the enzyme activity were 9.12,10.29 U/g respectively. This study preliminary confirmed that MCAD and LCAD had highly expression in liver and heart of Cyprinus carpio,similar as in Mammals,and high unsaturated fatty acid could promote the expression of LCAD.