Characterization and expression of gene encoding HMGR from Tripterygium wilfordii

【Objective】 This study aimed to clone the gene encoding HMGR,the rate-limiting enzyme in the plant terpenoid biosynthetic pathway from Tripterygium wilfordii,analyze its expression in suspension cell induced by chitosan with different concentrations,and improve the understanding of biosynthesis of terpenoid alkaloids in T.wilfordii.【Method】 The degenerate primers were synthesized based on the encoding sequence of the rate-limiting enzyme HMGR reported in GenBank and full-length hmgr cDNA sequence in the leaves of T.wilfordii was cloned by RT-PCR,5′-RACE and 3′-RACE.Bioinformatics analysis was used to identify the rate-limiting enzyme structure and functions of HMGR.Semi-quantitative RT-PCR was carried out to compare the expression of the hmgr gene in suspension cell and HPLC was used to determine the content of three secondary metabolites(triptolide,wilforgine and wilforine) after being induced by oligosaccharide with different concentrations of oligosaccharide.【Result】 A full-length cDNA encoding HMGR with a ORF of 1 860 bp and a total of 579 amino acids was obtained.Bioinformatics analysis revealed that the deduced HMGR had extensive homology with HMGRs of other plants.The molecular weight of the protein was 61.678 ku,the isoelectric point of HMGR was 6.98 and the stability index was 41.33.The protein was unstable.This protein contained two NADP(H)-binding sites(DAMGMNM and VGTVGGGT) and two HMG-CoA-binding sites(EMPVGYVQIP and TTEGCLVA).Gene expression by semi-quantative RT-PCR showed that the highest HMGR expression occured in T.wilfordii induced for 12 hours by oligosaccharide with a concentration of 50 mg/L.This concentration also played significant role in accumulation of triptolide,wilforgine and wilforine.【Conclusion】 HMGR in T.wilfordii may be the rate-limiting enzyme in MVA pathway and the gene encoding HMGR can stimulate the biosynthesis of terpenoids.