CN: 32-1845/R
ISSN: 2095-6975
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TONG Yu-Ru, ZHANG Yi-Feng, ZHAO Yu-Jun, HU Tian-Yuan, WANG Jia-Dian, HUANG Lu-Qi, GAO Wei. Differential expression of the TwHMGS gene and its effect on triptolide biosynthesis in Tripterygium wilfordii[J]. Chinese Journal of Natural Medicines, 2019, 17(8): 575-584

Differential expression of the TwHMGS gene and its effect on triptolide biosynthesis in Tripterygium wilfordii

TONG Yu-Ru1,2,3, ZHANG Yi-Feng3,4, ZHAO Yu-Jun2, HU Tian-Yuan3,4, WANG Jia-Dian3,4, HUANG Lu-Qi1,2, GAO Wei3,4,5
1 School of Pharmacy Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, China;
2 National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China;
3 School of Pharmaceutical Science, Capital Medical University, Beijing 100069, China;
4 School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069, China;
5 Advanced Innovation Center for Human Brain Protection, Capital Medical University, Beijing 100069, China
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.
Key words:    Overexpression    RNAi    HMGS    Triptolide    Acetyl-CoA   
Received: 2019-04-22   Revised:
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