Molecular cloning, expression and immunolocalization analysis of diphosphomevalonate decarboxylase involved in terpenoid biosynthesis from Euphorbia helioscopia L.

Abstract

ABSTRACTEuphorbia helioscopia L. is an herbaceous species of Euphorbia (Euphorbiaceae). As an ancient folk herbal medicine, the most effective medicinal component is terpenoid. Diphosphomevalonate decarboxylase (MDC) is the last rate-limiting enzyme of generating the isopentenyl pyrophosphate precursor of terpenoid in MVA pathway. The gene of MDC was cloned successfully from E. helioscopia, and named EhMDC (accession number: KP995936). The full length cDNA of EhMDC was 1653 bp, and it contained an open reading frame of 1245 bp, a 5′ untranslated region of 184 bp and a 3′ untranslated region of 224 bp. EhMDC contained 415 amino acids. Homologous sequence analysis showed that amino acid sequence of EhMDC had the highest identity of 88% with Ricinus communis MDC. Phylogenetic analysis of EhMDC indicated E. helioscopia, Hevea brasiliensis, R. communis, and Jatropha carcas which all belonged to Euphorbiaceae were classified into one class. Real-time PCR assay demonstrated EhMDC was constitutively expressed in roots, stems and leaves with a similar transcription level. Furthermore, in combination with immunoblot analysis and transmission electron microscopy immunogold labeling after anti-EhMDC antibody preparation, the EhMDC was found to be located in the cisternae of the endoplasmic reticulum, small vacuoles from endoplasmic reticulum and cytoplasm of laticifers. As a result, terpenoid biosynthesis site and accumulation of E. helioscopia laticifers were speculated.