ABSTRACT
3-Hydroxy-3-methylglutaryl-CoA synthase (HMGS) is the first committed enzyme in the MVA pathway and involved in the biosynthesis of terpenes in Tripterygium wilfordii. The full-length cDNA and a 515 bp RNAi target fragment of TwHMGS were ligated into the pH7WG2D and pK7GWIWG2D vectors to respectively overexpress and silence, TwHMGS was overexpressed and silenced in T. wilfordii suspension cells using biolistic-gun mediated transformation, which resulted in 2-fold increase and a drop to 70% in the expression level compared to cells with empty vector controls. During TwHMGS overexpression, the expression of TwHMGR, TwDXR and TwTPS7v2 was significantly upregulated to the control. In the RNAi group, the expression of TwHMGR, TwDXS, TwDXR and TwMCT visibly displayed downregulation to the control. The cells with TwHMGS overexpressed produced twice higher than the control value. These results proved that differential expression of TwHMGS determined the production of triptolide in T. wilfordii and laterally caused different trends of relative gene expression in the terpene biosynthetic pathway. Finally, the substrate acetyl-CoA was docked into the active site of TwHMGS, suggesting the key residues including His247, Lys256 and Arg296 undergo electrostatic or H-bond interactions with acetyl-CoA.
ABSTRACT
Tripterygium wilfordii 3-hydroxy-3-methylglutaryl coenzyme-A reductase (TwHMGR) is an important regulation site in terpenoids metabolic pathway in cytoplasm which is the first speed limit enzyme of MVA pathway. In order to investigate the effects of TwHMGR on the biosynthesis of triptolide and celastrol in Tripterygium wilfordii, the overexpression of TwHMGR (OE-HMGR) was studied in this paper. We cloned the full-length of TwHMGR to construct overexpression vector by Gateway technology then delivered the expression vector into Tripterygium wilfordii suspension cells by gene gun. qRT-PCR was used to detect the expression of TwHMGR:the expression of TwHMGR was increased to 1.75 folds over the control group (empty vector:pH7WG2D) in the overexpression 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 to 163.93% and 190.04% of over the control group in the overexpression group. Based on these findings, the positive effect on the accumulation of active terpenoids, triptolide and celastrol in Tripterygium wilfordii was found and the results laid a foundation of the synthetic biology research on important active terpenoids in Tripterygium wilfordii.
ABSTRACT
MCT is an important key enzyme in the terpenoid biosynthesis in MEP pathway. In this study, Gateway technology was used to construct RNAi vector of TwMCT, and a vector fragment with a size of 484 bp was obtained. The TwMCT RNAi vector was transferred into the suspension cells of Tripterygium wilfordii by gene gun. Accumulation of terpenoids was assayed by UPLC, and the result showed that the content of triptolide and celastrol in cells decreased by 23.4% and 42.8%, respectively, compared with the control group pK7GWIWG2D. Moreover, the gene expression of TwMCT and major genes in terpenoid biosynthesis pathway was detected by qRT-PCR, which demonstrated that the expression of TwMCT reduced by 29.2% relative to that of the control group pK7GWIWG2D, and the relative expression of TwDXR, TwGGPS, TwHMGR and TwHMGS diminished by 36.3%, 31.3%, 62.2%, and 29.1%, respectively, but the expression of TwDXS was up-regulated by 114.2%, and there was no significant change in TwFPS. Thus, it was verified in vivo that interference with TwMCT expression significantly inhibited the accumulation of triptolide and celastrol in Tripterygium wilfordii, laying a foundation for further exploring the regulation mechanism of MCT gene on the terpenoid biosynthesis in Tripterygium wilfordii.