Transcriptome analysis identifies novel responses and potential regulatory genes involved in 12-deoxyphorbol-13-phenylacetate biosynthesis of Euphorbia resinifera

Abstract

12-Deoxyphorbol-13-phenylacetate (DPP), found in Euphorbia resinifera, is a prototype of a new class of phorbol derivatives that function as protein kinase C activators with potent anti-tumor-promoting activity. To better understand the biosynthesis of DPP in E. resinifera and develop methods for its production, we used next-generation sequencing technologies to build a transcriptome dataset. We obtained a total of approximately 27,102,078 clean reads that were assembled into 90,448 unigenes. Of those, 129 and 59 unigenes were identified in the Terpenoid Backbone Biosynthesis (TBB) (KEGG map00900) and the Diterpenoid Biosynthesis (DB) (KEGG map00904) pathways, respectively. We investigated the expression patterns of 23 DPP biosynthesis-related genes and validated the results by quantitative real time PCR. The results showed that CMK (4-diphosphocytidyl-2-C-methyl-d-erythritol kinase), MECS (2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase), HDS (1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase), and IPI (Isopentenyl-diphosphate Delta-isomerase) of the methylerythritol 4-phosphate (MEP) pathway had a higher expression compared to other genes of TBB, whereas the expression levels of genes involved in the mevalonic acid (MVA) pathway were relatively lower and not significantly different from each other. Furthermore, the expression of the 23 genes in root, stem and young stem differed, and the statistical results indicated that HMGS (Hydroxymethylglutaryl-CoA synthase), MVK (mevalonic acid), MECT (2-C-methyl-d-erythritol 4-phosphate cytidylyltransferase), MECS, HDS and IPI in the TBB pathway as well as KS in the DB pathway were significantly and positively correlated with the CS (casbene synthase) gene. These results suggested that both cytosol-localized MVA and plastid-localized MEP pathways may have a significant contribution in DPP accumulation, with MEP pathway playing a dominant role. To the best of our knowledge, this study is the first transcriptome analysis of E. resinifera and the results reported here will aid the identification of functional genes and better understanding of DPP biosynthesis.