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

doi:10.7668/hbnxb.2017.03.008

Abstract

Abstract: 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.

Key words: Cyprinus carpio, MCAD, LCAD, Clone, Prokaryotic expression, Enzyme activity

CLC Number:

YU Wenjuan, YU Juhua, LI Hongxia, LI Jianlin, TANG Yongkai, YU Fan, HE Feng, FU Chunjie. Cloning, Expression and Activity Analysis of Two Kinds ACADs in Cyprinus carpio[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2017, 32(3): 48-57. doi: 10.7668/hbnxb.2017.03.008.

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