Abstract
Triptolide, an important bioactive diterpenoid extracted from the plant Tripterygium wilfordii, exhibits many pharmacological activities. MYC2 transcription factor (TF) plays an important role in the regulation of various secondary metabolites in plants. However, whether MYC2 TF could regulate the biosynthesis of triptolide in T. wilfordii is still unknown. In this study, two homologous MYC2 TF genes, TwMYC2a and TwMYC2b, were isolated from T. wilfordii hairy roots and functionally characterized. The analyses of the phylogenetic tree and subcellular localization showed that they were grouped into the IIIe clade of the bHLH superfamily with other functional MYC2 proteins and localized in the nucleus. Furthermore, yeast one-hybrid and GUS transactivation assays suggested that TwMYC2a and TwMYC2b inhibited the promoter activity of the miltiradiene synthase genes, TwTPS27a and TwTPS27b, by binding to the E-box (CACATG) and T/G-box (CACGTT) motifs in their promoters. Transgenic results revealed that RNA interference of TwMYC2a/b significantly enhanced the triptolide accumulation in hairy roots and liquid medium by upregulating the expression of several key biosynthetic genes, including TwMS (TwTPS27a/b), TwCPS (TwTPS7/9), TwDXR, and TwHMGR1. In summary, our findings show that TwMYC2a and TwMYC2b act as two negative regulators of triptolide biosynthesis in T. wilfordii hairy roots and also provide new insights on metabolic engineering of triptolide in the future.