Expression,Purification And Characterization Of Four Sesquiterpene Synthases From Salvia Miltiorrhiza

Salvia miltiorrhiza Bunge, a famous traditional Chinese medicine, is a perennial plant in the genus Salvia of the Lamiaceae family. Generally, its root or rhizome is the main material to medicine. Research of modern chemistry and pharmacology shows that S. miltiorrhiza is apparent effect to treat and prevent cardiovascular, cerebrovascular, hyperlipidemia. The study of volatile terpenoids in S. miltiorrhiza is rare. And the research of terpenoid biosynthetic pathway is needed to discovery. This study adopted the technology of RACE to cloning four sesquiterpene synthase genes, YL-1、YL-4、YL-5 and YL-6, and carried on the prokaryotic expression in E.coli, detected enzyme activity in vitro. Then protein purification and detect enzyme kinetics parameters. The main findings were:1.Full length cDNA sequences for YL-1、YL-4、YL-5 and YL-6 genes were separated from S. miltiorrhiza. The obtained YL-1 cDNA contained 1629bp and encoding 542 amino acid (AA) residues, the obtained YL-4 cDNA contained 1641bp and encoding 546 amino acid (AA) residues, the obtained YL-5 cDNA contained 1641 bp and encoding 546 amino acid (AA) residues and the obtained YL-4 cDNA contained 1641 bp and encoding 546 amino acid (AA) residues. Blast result showed that the separated cDNAs have very high homogy with the sesquiterpene synthase genes that have been identified.2. The pET28a was chosen as the expression vector of 4 genes to construct recombinant plasmid and expressed in Escherichia coli. Obtained the recombinant proteins after SDS-PAGE analysis and detected enzyme activity. The four major products formed by recombinant YL-1 enzyme activity with farnesyl diphosphate(FPP) as substrate are a-bergamotene-. a-santalene> (E)-(3-farnesene and p-Sesquiphellandrene; And the only product formed by recombinant YL-1 enzyme activity with FPP as substrate is valencene. Using purified protein measured enzyme kinetic parameters, the result showed Km of YL-1 is 16.47 μM and the kcat value of YL-1 is 0.2268 min-1; And the Km of YL-4、YL-5 and YL-6 are 73.34 μM,21.53 μM and 17.39 μM, the kcat value of YL-4、YL-5 and YL-6 are 297.7 min-1、128.8 min-1 and 111.9min-1Using Actin as reference gene, analysis the expression levels of YL-1, YL-4, YL-5 and YL-6 in different positions of S. miltiorrhiza by real-time PCR. Expression of YL-1 in the descending order is the root> leaf> flower> stem. Expression of YL-4 and YL-5 in the descending order is the leaf> flower> root> stem. Expression of YL-6 in the descending order is the flower> leaf> root> stem.4 h after the application of exogenous hormone, we isolated RNA of aerial organs and analyzed the variation of 4 genes expression. Ethylene and abscisic acid on 4 genes expression level showed positive regulation. Salicylic acid made the relative expresstion of YL-1、YL-4、YL-5 and YL-6 lower and the effect is obvious.