MYC2 Transcription Factors TwMYC2a and TwMYC2b Negatively Regulate Triptolide Biosynthesis in Tripterygium wilfordii Hairy Roots

Abstract

Triptolide, an important bioactive diterpenoid extracted from the plant <i>Tripterygium wilfordii</i>, 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 <i>T. wilfordii</i> is still unknown. In this study, two homologous MYC2 TF genes, <i>TwMYC2a</i> and <i>TwMYC2b</i>, were isolated from <i>T. wilfordii</i> 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, <i>TwTPS27a</i> and <i>TwTPS27b</i>, by binding to the E-box (CACATG) and T/G-box (CACGTT) motifs in their promoters. Transgenic results revealed that RNA interference of <i>TwMYC2a/b</i> significantly enhanced the triptolide accumulation in hairy roots and liquid medium by upregulating the expression of several key biosynthetic genes, including <i>TwMS</i> (<i>TwTPS27a/b</i>), <i>TwCPS</i> (<i>TwTPS7/9</i>), <i>TwDXR</i>, and <i>TwHMGR1</i>. In summary, our findings show that TwMYC2a and TwMYC2b act as two negative regulators of triptolide biosynthesis in <i>T. wilfordii</i> hairy roots and also provide new insights on metabolic engineering of triptolide in the future.