A New 1-Deoxy-D-xylulose 5-Phosphate Reductoisomerase Gene Encoding the Committed-Step Enzyme in the MEP Pathway from Rauvolfia verticillata

Abstract

1-Deoxy-d-xylulose 5-phosphate (DXP) reductoisomerase (DXR; EC 1.1.1.267) catalyzes a committed step of the methylerythritol phosphate (MEP) pathway for the biosynthesis of pharmaceutical terpenoid indole alkaloid (TIA) precursors. The full-length cDNA sequence was cloned and characterized from a TIA-producing species, Rauvolfia verticillata, using rapid amplification of cDNA ends (RACE) technique. The new cDNA was named as RvDXR and submitted to GenBank " to be assigned with an accession number (DQ779286). The fulllength cDNA of RvDXR was 1804 bp containing a 1425 bp open reading frame (ORF) encoding a polypeptide of 474 amino acids with a calculated molecular mass of 51.3 kDa and an isoelectric point of 5.88. Comparative and bioinformatic analyses revealed that RvDXR showed extensive homology with DXRs from other plant species and contained a conserved transit peptide for plastids, an extended Pro-rich region and a highly conserved NADPHbinding motif in its N-terminal region owned by all plant DXRs. The phylogenetic analysis revealed that DXRs had two groups including a plant and bacterial group; RvDXR belonged to angiosperm DXRs that were obtained from Synechocystis through gene transfer according to the phylogenetic analysis. The structural modeling of RvDXR showed that RvDXR had the typical V-shaped structure of DXR proteins. The tissue expression pattern analysis indicated that RvDXR expressed in all tissues including roots, stems, leaves, fruits and followers but at different levels. The lowest transcription level was observed in followers and the highest transcription was found in fruits of R. verticillata; the transcription level of RvDXR was a little higher in roots and stems than in leaves. The cloning and characterization of RvDXR will be helpful to understand more about the role of DXR involved in R. verticillata TIA biosynthesis at the molecular level and provides a candidate gene for metabolic engineering of the TIAs pathway in R. verticillata.

Molecular characterization and expression of 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) gene from Salvia miltiorrhiza

“…3). Therefore, SmDXR was considered to be a constitutively expressing gene, which is similar with the most DXRs such as Camptotheca acuminata (Yao et al 2008), Rauvolfia verticillata (Liao et al 2007), Ginkgo biloba (Gong et al 2005) and Hevea brasiliensis (Yoonram et al 2008). This is the first report on the mRNA expression profile of genes encoding key enzymes involved in tanshinones biosynthetic pathway in different tissues of S. miltiorrhiza plants.…”

Reference Genes for the Normalization of Gene Expression in Eucalyptus Species

“…The MEP pathway, through which diterpenes are synthesized, has two important initial steps: (i) the formation of 1-deoxy-D-xylulose 5-phosphate (DXP) from pyruvate and glyceraldehyde 3-phosphate through the action of the DXP synthase (DXS), followed by the conversion of DXP into MEP by the action of the DXP reductoisomerase (DXR). As DXS and DXR are key enzymes catalyzing the two committed steps for isoprenoid biosynthesis, genes coding for DXS and DXR may play important roles in controlling the plastidic synthesis of isoprenoids and the downstream diterpene products (Carretero-Paulet et al 2002, Liao et al 2007, Wu et al 2009, Yan et al 2009.…”

“…Based on the Eucalyptus sequences, primers were designed as shown along with amplicon lengths (bp). Eucalyptus reference genes (continued) higher levels of dxr mRNA were reported in fruits and roots, with the lowest levels in flowers (Liao et al 2007). In order to confirm the constitutive expression of the three best Eucalyptus genes selected as references (Eucons04, Eucons08 and Eucons21), we tested them by normalizing the patterns of dxr gene expression in Eucalyptus and compared the results with those normalized by the traditional reference genes RibL23A and GAPDH.…”

Molecular cloning and characterization of the gene encoding 2-C-methyl-D-erythritol 4-phosphate cytidyltransferase from hairy roots of Rauvolfia verticillata

“…The third reaction (the formation of 4-diphosphocytidyl-2C-methyld-erythritol from MEP and CTP) in the MEP pathway is catalyzed by 2-C-methyl-D-erythritol 4-phosphate cytidyltransferase (MCT) (Gabrielsen et al 2006). In order to map the biosynthetic pathway of ajmalicine in R. verticellata at the molecular level, our group reported four MEP-pathway genes including 1-deoxy-D-xylulose5-phosphate reductoisomerase (DXR) (Liao et al 2007), 2-C-methyl-D-erythritol Table 1. The primers used for the real-time Q-PCR.…”