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

摘要

The aim of this study was to verify the effects ofTwIDI(GenBank: KT279355.1) on triptolide and celastrol accumulation in the biosynthesis of terpenoids inTripterygium wilfordiiand the regulation of the expression of related genes in the triptolide and celastrol biosynthesis pathway. After bioinformatics analysis ofTwIDI, 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 intoTripterygium wilfordiisuspension cells. qRT-PCR was used to detect the expression ofTwIDIand related genes in the triptolide and celastrol biosynthesis pathway. The expression ofTwIDIwas 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,TwMCSwas reduced to 62% of the control whenTwIDIwas overexpressed and increased to 188% in the RNAi group. The expression ofTwDXSdid not change significantly both duringTwIDIoverexpression and RNAi group. The accumulation of triptolide and celastrol in the suspension cells ofTripterygium wilfordiiwas 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 inTripterygium wilfordiiand the feedback regulation of genes in the triptolide and celastrol biosynthesis pathway was verified throughTwIDIoverexpression and RNAi experiments.