The expression of TwDXS in the MEP pathway specifically affects the accumulation of triptolide.

1-Deoxy-d-xylulose-5-phosphate synthase (DXS) is the first enzyme in the plant 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway of terpenoid synthesis. TwDXS is a prominent protein in the Tripterygium wilfordii proteome, with especially high expression in the root periderm. It is significantly regulated by methyl jasmonate. Here, we studied the influence of TwDXS expression on bioactive terpenoids in T. wilfordii. Specific fragments of TwDXS (GenBank: AKP20998.1) with lengths of 2148 and 437 bp were amplified to construct the overexpression (OE) and RNA-interference (RNAi) vectors, respectively. After transformation of suspension cells, the expression of TwDXS and genes related to the terpenoid biosynthetic pathway was measured using qRT-PCR. TwDXS mRNA level was 153 and 43% of the control in the OE and RNAi lines. Related genes in the 2-C-methyl-d-erythritol 4-phosphate (MEP), mevalonic acid (MVA) and downstream pathways showed similar trends to the changes of TwDXS expression. Ultra Performance Liquid Chromatography (UPLC) was employed to measure the accumulation of terpenoids. Importantly, the triptolide content showed significant differences in both the TwDXS OE (222.35% of the control) and RNAi (34.86% of the control). However, there were no obvious changes in the celastrol content. In this study, we verified that the expression of TwDXS affects triptolide but not celastrol in T. wilfordii via both TwDXS OE and RNAi experiments.