Discovery Of Sesquiterpenes Inducer And Cloning,Gene Expression Of Terpenoids Synthase Gene From Aquilaria Sinensis

Agarwood is the black resinous portions from xylogen in the tree of Aquilaria sinensis(Lour.)Gilg.Aquilaria sinensis is the unique resource of agarwood in China.Under the normal circumstances,it grows healthily and the xylem is white without fragrant.The agarwood produced when the Aquilaria species exposed to the external conditions,such as wind,thunder strike,chopped and insects.The main chemical compounds of agarwood are sesquiterpenes,phenylethyl chromone derivatives and aromatic.The chemical components of artificial agarwood differ greatly to natural agarwood,the content of sesquiterpenes and phenylethyl chromone derivatives are equaly in natural agarwood,while the mainly component of artificial agarwood is phenylethyl chromone derivatives.Sesquiterpenes is considered to be defensive substances from A.sinensis in the face of adversity stress;it is also the main effective component and characteristic constituent.The selection of sesquiterpenes inducer and rate-limiting enzyme genes cloning,expression analysis in terpenoids synthetic route,which have important significance to illustrate the molecular mechanism of agarwood,regulate the artificial inducer process and improve the quality of artificial agarwood.The previous experiment results show that the formic acid plus endophytic fungi A13 has the best effect on promote A.sinensis product resin.On this basis,the model of A.sinensis excised twig was used to choose the inducer,which can format sesquiterpene components efficiency and specificity.A.sinensis excised twig inoculated with formic acid,salicylic acid,methyl jasmonate and endophytic fungi A13.GC-MS profiling was employed to research the metabolites of excised twig.Meanwhile,we analyzed the expression of several genes from A.sinensis using Fluorescence-based quantitative real-time PCR(qPCR).Combining the result of GC-MS and qPCR,we can evaluate the effects of inducers comprehensively.The inducer groups of Methyl jasmonate of 1% concentration plus A13 of 10% and Methyl jasmonate of 1% concentration plus A13 of 15% concentration can increase the production of sesquiterpenes.HMGR is the rate-limiting enzyme genes in terpenoids synthetic route,which were obtained from cDNA of A.sinensis based on previous transcriptome sequencing data.The HMGR sequence contained an ORF of 1799 bp.The gene was inserted into Prokaryotic expression vector pET22 b,then succefully expressed in Escherichia coli,and used affinity chromatography and gel to purify the product.According to the result of SDS-PAGE and Western-blot,the protein 3-hydroxy-3-methyl-glutaryl-CoA(HMGR)reductase was expressed in the form of inclusion bodies with a molecular weight of 68 kD.The optimal induction condition of HMGR protein is 0.5 mM IPTG,37 ?,4 h.The terpenoids synthases HMGR catalyzed the substrate 3-hydroxy-3-methylglutaryl coenzyme A(HMG-CoA),and the optimal enzymatic reaction condition is 0.1 mM HMG-CoA,37 ?,pH 7.0 with reaction time of 4 min.This study combines the analysis of metabolites and expression of several terpenoids synthases genes to evaluate the results of inducer.Cloning and expression of terpenoids synthase gene lay the foundation of explain the sesquiterpene biosynthesis mechanism and improve the content of sesquiterpene in A.sinensis.The study provides a scientific basis for metabolism controlling in A.sinensis and artificial induction method optimization.