Cloning Of Genes Encoding HMGR From Tripterygium Wilfordii And Its Metabolic Regmlation

Tripterygium Wilfordii as one of tranditional Chinese medicines, has powerfulinsecticidal effect and high medical value. For the complex chemical constituents and the lowcomponent of some active substances, the main active compounds couldn't be extracted orsynthesized by chemical process in abundance. And, as overexploitation and weak sense ofnatural resources protection, Tripterygium Wilfordii with exhausted resourses is limited inpractical use.Therefore, a study was made on biosynthesis of secondary terpenoid metabolitefrom Tripterygium Wilfordii. The full-length cDNA encoding3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) was cloned by RACE from Tripterygium Wilfordii. The full-lengthhmgr cDNA was1909bp, containing98bp5' untranslated region, a3' untranslated region of50bp, poly A tail and a1860bp open reading frame that encodes a peptide of579amino acids.Bioinformatic analyses revealed that the isoelectric point of HMGR is6.98, and themolecular weight of protein is61.678KDa. The instability index is computed to be41.33, andthe protein is classified as unstable. This protein contained two transmembrane domains,localized in A29~A51and A71~A93; and two putative HMGR binding sites(EMPVGYVQIP andTTEGCLVA), the G amino acid residue had an important promotion; besides, there were twoNADP(H)-binding sites(DAMGMNM and VGTVGGGT). The secondary structure analysisshowed that HMGR was comprised of43.35％alpha helix,17.10％extended strands,5.01%beta turn and34.54%random coil. The three dimensional structure of HMGR was "V"model.The deduced amino acid sequence of HMGR from Tripterygium Wilfordii exhibits74%-80%homoly with other plant HMGRs, and the phylogenetic tree analysis indicated that HMGRfrom Tripterygium Wilfordii belongs to the plant HMGR family and it has the closetrelationship with HMGRs from Dimocarpus longan and Litchi chninese. Besides, hmgrexpressed highest in leaf, and followed by root and suspicion cell.Based on HPLC and RT-PCR, the relationship between the expression of HMGRand the accumulation of wilforgine, wilforine and triptolide in Tripterygium Wilfordiisuspicion cells induced by chitosan had been analyzed. Expression analysisi showed thatHMGR from Tripterygium Wilfordii expressed higher in suspicion cells which tested by50 mg/L chitosan after12hours. In addition, the results indicated that the terpenoids, such aswilforgine, wilforine and triptolide, accumulation maybe resulted from the co-expressionup-regulation of HMGR, which encoded the first related enzyme in MVA pathway.