Cloning and Expression Analysis of DXS Gene from Pogostemon cablin(Blanco)Benth.
1-Deoxy-D-xylulose-5-phosphate synthase(DXS)is the first key enzyme to regulate the MEP pathway in the terpene synthesis pathway.In order to illustrate the molecular mechanism of DXS gene partici-pating in synthesis and regulation of the main terpenoid component,patchouli alcohol,in patchouli(Pogoste-mon cablin),the research was conducted based on the transcriptome DXS gene sequence obtained previously by our research group.PcDXS gene was cloned by using patchouli cDNA as template,and then analyzed by bioinformatics method.The prokaryotic expression vector pET-28a-PcDXS was constructed to induce protein expression,and real-time fluorescence quantitative PCR was used to detect the expression of DXS gene in roots,stems,leaves and buds of patchouli.The results showed that PcDXS1 and PcDXS2 gene sequences with open reading frame length of 2 151 bp and 1 908 bp were cloned from leaves of patchouli,encoding 716 and 635 amino acids,respectively.PcDXS1 protein was predicted to have 78.33 kDa of molecular weight,and was a stable,non-transmembrane and non-secreted protein primarily localized in chloroplasts.PcDXS2 protein had 67.92 kDa of molecular weight,which was an unstable,non-transmembrane and non-secreted protein mainly localized in chloroplasts.PcDXS1 and PcDXS2 contained DXP̠synthase̠N,Transket̠pyr and Transketolase̠C domain modules respectively,belonging to DXS superfamily.PcDXS1 and PcDXS2 had high homology in gene sequence with DXS genes of Scutellaria barbata and Phlomis umbrosa.PcDXS1 and PcDXS2 fusion proteins induced by isopropyl-β-D-thiogalactoside(IPTG)were mainly expressed in precipitation.The expression lev-els of PcDXS1 and PcDXS2 genes were the lowest in roots.PcDXS1 gene was significantly higher expressed in leaves,while PcDXS2 gene was relatively higher in leaves,buds and stems compared to roots.The results could lay foundations for further studies on biological functions of DXS gene in terpenoid compound synthesis pathway and gene regulation mechanism of terpenoid metabolism pathway in patchouli.
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