Overexpression and RNAi-mediated downregulation of TwIDI regulates triptolide and celastrol accumulation in Tripterygium wilfordii

The aim of this study was to verify the effects of TwIDI (GenBank: KT279355.1) on triptolide and celastrol accumulation in the biosynthesis of terpenoids in Tripterygium wilfordii and the regulation of the expression of related genes in the triptolide and celastrol biosynthesis pathway. After bioinformatics analysis of TwIDI, we cloned the full-length CDS and a specific 398 bp fragment to construct overexpression and RNAi vectors, respectively. The specific amplification of hygromycin and kanamycin resistance gene fragments confirmed that the expression vectors had been successfully delivered into Tripterygium wilfordii suspension cells. qRT-PCR was used to detect the expression of TwIDI and related genes in the triptolide and celastrol biosynthesis pathway. The expression of TwIDI was increased to 157% of the control group (empty vector) in the overexpression group, and was reduced to 71% of the control group in the RNAi group. Notably, the expression of other genes in the triptolide and celastrol biosynthesis pathway also showed differences. For example, TwMCS was reduced to 62% of the control when TwIDI was overexpressed and increased to 188% in the RNAi group. The expression of TwDXS did not change significantly both during TwIDI overexpression and RNAi group. The accumulation of triptolide and celastrol in the suspension cells of Tripterygium wilfordii was detected by UPLC, revealing that the contents of triptolide and celastrol were increased 1.36- and 1.20-fold over the control group in the overexpression group, and decreased to 0.16 and 0.36 of the control group in the RNAi group. Based on these findings, the effect on the accumulation of active terpenoids in Tripterygium wilfordii and the feedback regulation of genes in the triptolide and celastrol biosynthesis pathway was verified through TwIDI overexpression and RNAi experiments.