| Salvia miltiorrhiza Bunge is often studied as an important medicinal plant.Its dried roots and rhizomes are used as medicine to treat cardiovascular and cerebrovascular diseases,antioxidation and antitumor.Most researchers nowadays focuse on the study of diterpenoid tanshinones as a secondary metabolite in S.miltiorrhiza.However,little research has been conducted on triterpenoids in S.miltiorrhiza.Since many triterpenoids as medicinal active ingredients are important,and the metabolic pathway of triterpenoids are analyzed and studied.Here,we based on the transcriptome database of the S.miltiorrhiza,which was established in previous study,obtained a unigene of 2,3-oxidosqualene cyclase(SmOSC),and used RACE technology to clone the full-length cDNA of SmOSC.The main results are as follows:1.A 2,3-oxidosqualene cyclase(SmOSC)was cloned from S.miltiorrhiza.After sequence alignment,the full-length 2298 bp gene was identified and encoded a total of 765 amino acid residues.This gene may encode a 2,3-oxidosqualene cyclase.Subsequent subcellular localization of SmOSC by PSORT online server,prediction and analysis of physicochemical properties of protein on Protparam,online software of ExPASy Proteomics Server,prediction of secondary structure of protein-encoded online using predictor protein,and use the biological software Swiss-model to predict the three-dimensional structure of the protein and to establish a model.It was found that the SmOSC has a high homology with the triterpene cyclase PgDDS.2.Through Gateway’s method,the SmOSC gene was firstly transferred into the entry vector pDonr207,and then it was subjected to LR reaction,recombined into the pAg1423 yeast expression vector,and then transformed to the Y21900 yeast mutant strain.Through the self-fermentation in the yeast cell,the in vivo enzyme activity reaction is performed,and the yeast product is extracted for detection and analysis.The compound was analyzed on a Q-Exactive(QE)mass spectrometer and the results showed that the produc was damethylene glycol(DM Ⅱ).3.Studies using microorganisms as the chasis are more common.In this study,based on yeast cell studies,plant cell chasis was also considered.The model plant Nicotiana benthamiana was ideally studied for its short growth cycle.Six-week-old N.benthamiana grown in the greenhouse was selected for transient expression.The Gateway method was adoptted to construct the expression vector pEAQ-SMOSC,and then the expression vector was introduced into Agrobacterium(strain GV3101).For transient transformation,Agrobacterium cells harboring pEAQ-SmOSC was infiltrated into the abaxial side of the leaves of N.benthamiana using a syringe.After 4 days of culture,the infected areas were extracted and the chemicals were detected by QE.The results showed that the produc of transient expression by N.benthamiana was DM Ⅱ.4.Using real-time fluorescence quantitative PCR(Real-time PCR)technology,we studied the difference of gene expression of SmOSC in roots,stems,leaves and flowers of S.miltiorrhiza.First,RNA was extracted from different tissues,and Actin was used as internal reference gene.The results showed that the expression patten of SmOSC in S.miltiorrhiza was flower>leaf>stem>root.That is,the expression level of SmOSC in the overgrand parts of S.miltiorrhiza were higher.Subsequently,we carried out a hormone induced expression experiment,that is,exogenous hormone jasmonate(MJ),ethylene(ET),gibberellic acid(GA),abscisic acid(ABA)and salicylic acid(SA)were applied to S.miltiorrhiza.After 2 h,RNA was extracted from leaves and real-time PCR analysis of SmOSC was carried out.The results showed that the expression of SmOSC was down regulated under the treatments of ABA,MJ,SA and GA.Compared with the Actin expression,the expression of SmOSC was only slightly decreased by 0.1 under ET treatment.The results demonstrated that the expression of SmOSC was specilized in different tissues and was regulated by tress environment. |
