Molecular Cloning and Characterization of Two Key Enzymes involved inthe Diterpenoid Biosynthesis Pathway of Isodon rubescens

Abstract

Isodon rubescens, an important medical plant, contains various terpenoids. This plant’s active compounds are primarily oridonin with antitumor properties. As the precursor for oridonin biosynthesis, are synthesized by MEP pathway. On the basis of our earlier studies, we isolated and cloned two important genes catalyzing diterpenoid biosynthesis in the MEP pathway. 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase and 4-hydroxy-3- methylbut-2-enyl diphosphate reductase are the fifth enzymes and the last step key enzyme for the methylerythritol phosphate (MEP) pathway, respectively, which is important for the regulation of isoprenoid biosynthesis. Sequence analysis revealed that DcIspF (accession no. KT948057) was 966 bp, contains a gene open reading frame (ORF) of 708 bp belonging to the MECDP-synthase superfamily and DcIspH (accession no KT948058) contains a 1389 bp ORF encoding a predicted 462 amino acid polypeptides as a member of the lytB_ispH superfamily. The deduced DcIspF and DcIspH amino acid sequences shared high similarity with DcIspF and DcIspH of other plant respectively, each of them exhibiting an N-terminal transit peptide and conserved amino acid sites. Quantitative real-time PCR analysis showed that the expression of DcIspF was considerably higher in leaves, the lowest in callus. These results indicate that we have identified functional DcIspF and DcIspH enzymes, which may play a pivotal role in the biosynthesis of diterpenoid in I. rubescens.