水芹OjDXS基因克隆、表达及酶活鉴定OA

1-Deoxy-D-xylulose-5-phosphate synthase(DXS)is the first key enzyme in the 2-C-methyl-D-erythritol 4-phosphate(MEP)pathway for terpenoid biosynthesis and plays an important role in the biosynthesis of terpenoids in Oe-nanthe javanica(Blume)DC.To determine the sequence characteristics,tissue expression pattern and enzymatic activity of the DXS gene in O.javanica,gene cloning,bioinformatic analysis,qRT-PCR,prokaryotic expression,protein purifi-cation etc.were used to conduct the preliminary functional characterization of OjDXS gene which was cloned from the O.ja-vanica Fuqin NO.1(FQ1H).Full sequence length of the open reading frame(ORF)of OjDXS gene was 2 139 bp,enco-ding 712 amino acids.The encoded protein contained several conserved domains,including the transketolase pyrimidine-binding domain(Transket_pyr),the C-terminal transketolase domain(Transketolase_C),and the DXP_synthase_N do-main.The amino acid sequences of DXS were highly conserved in different plants.According to the evolutionary relation-ships,OjDXS exhibited high phylogenetic relationship with DXS from Daucus carota subsp.sativus,which belonged to Apiaceae.The expression levels of the OjDXS gene in leaves was significantly higher than in other tissues.The enzyme activity of DXS in O.javanica was the highest in the leaves and the lowest in the roots.The prokaryotic expression vector pCold-OjDXS was constructed by homologous recombination technology and transformed into Escherichia coli for prokaryotic expression.The OjDXS protein was further purified and the DXS enzyme activity of purified OjDXS protein was 621.28±33.28 U·uL-1.The results of this study provide a theoretical reference for subsequent OjDXS gene function analysis.