草麻黄SDR基因克隆、表达及功能验证OA

To elucidate the biosynthetic pathway of amphetamine-type alkaloids in ephedra,this study cloned a Short-Chain Dehydrogenase/Reductase(SDR)gene from the herbaceous stems of Ephedra sinica,constructed it into the pET-32a vector,and transformed it into Escherichia coli DH5α.The recombinant plasmid was then introduced into E.coli Rosetta(DE3)for induction and purification of the SDR protein.The enzymatic products of SDR were analyzed by gas chromatography-mass spectrometry(GC-MS)to verify its reductase activity.The results showed that the cloned E.sinica SDR gene was 756 bp in length,encoding a protein of 251 amino acids with a relative molecular weight of 26.65 kDa.The protein belongs to the NADB-Rossmann superfamily,exhibits hydrophobicity,and contains no transmembrane domains.Phylogenetic analysis demonstrated that the SDR amino acid sequence from E.sinica shared the closest evolutionary relationship with that of Cryptomeria japonica.Through the prokaryotic expression system,the recombinant protein was efficiently expressed in E.coli.GC-MS analysis demonstrated that this recombinant protein catalyzed the conversion of 3,5-bis(trifluoromethyl)acetophenone into 1-(3,5-bis(trifluoromethyl)phenyl)ethan-1-ol in vitro.This study successfully cloned and heterologously expressed the SDR gene from E.sinica and confirmed its oxidoreductase activity.These findings provide a foundation for understanding the biosynthesis of amphetamine-type alkaloids in E.sinica and potential applications in genetic engineering.