Cloning Of Key Genes Of JA Signal Transduction Pathways And MYC2 Overexpression And Antisense RNA Silencing In Pogostemon Cablin

Pogostemon cablin（Blanco）Benth.is one of the traditional Chinese medicines,which used to treat dampness,vomiting,fever and fatigue.The main active ingredient of its volatile oil is sesquiterpene of patchouli alcohol,which has been widely used in medicine and food industries by its great medicinal and economic value in recent years.Jasmonic acid participate in many physiological activities of plants such as regulating the growth,responding to stress,and affecting the synthesis of secondary metabolites.JAZ/COI1/MYC2 is the core module of the JA signal transduction pathway.In recent years,studies have shown that MYC2 regulate the biosynthesis of sesquiterpenoids.MYC2 is the core transcription factor of the JA signal transduction pathway.Therefore,the study of the key genes of JA signal transduction pathways in Pogostemon cablin is of great significance for in-depth understanding of the molecular mechanism of patchouli secondary metabolism regulation.In this study,the expression levels of the key genes MYC2,JAZ2,COI1in the JA signal transduction pathway and PTS,FPPS,SQLE in the sesquiterpene synthesis pathway were analyzed after treatment by Me JA.Based on the patchouli transcriptome database of the laboratory,the patchouli MYC2,JAZ2,COI1,and JAR1 genes were cloned and analyzed by bioinformatics.Through heterologous expression in escherichia coli,the optimal conditions for IPTG to induce the expression of MYC2 protein were investigated,and soluble MYC2 protein was obtained.Constructed plant expression vectors of patchouli MYC2 gene overexpression（GBD）and antisense RNA silencing（GR）,and established a patchouli genetic transformation system mediated by agrobacterium tumefaciens GV3101.In addition,the expression level of sesquiterpene pathway genes DXS,DXR,MCT,CMK,MCS,HDS,HDR,AACT,HMGS,HMGR,MVK,PMK,MVD,GPPS,GGPPS,FPPS,PTS in patchouli roots,stems,and leaves were studied.The main results are as follows:1.Me JA promoted the expression of JAZ2,MYC2,COI1,PTS,FPPS,SQLE,and JAZ2、MYC2 are up-regulated extremely significantly.Different concentrations of Me JA have different effects on gene expression.JAZ2 is the main gene induced by Me JA in JA signal transduction pathway,which can activate the co-expression of FPPS gene in sesquiterpene synthesis pathway,and then affect the synthesis of sesquiterpene such as patchouli alcohol.2.The open reading frame（ORF）of patchouli MYC2 gene was 1338 bp,which encoded 446 amino acids.MYC2 protein mainly located in the nucleus,and was a non-secretory,non-transmembrane unstable protein,which belongs to the b HLH transcription factor family and has a high homology with Salvia miltiorrhiza MYC2.The ORF of the patchouli JAZ2gene was 948 bp,which encoded 316 amino acids.JAZ2 protein mainly located in the nucleus,and was a non-secretory,non-transmembrane unstable protein,which belongs to the TIFY superfamily.The ORF of patchouli COI1gene was 1821 bp,which encoded 607 amino acids.COI1 protein mainly located in the chloroplast,and was a non-secretory,non-transmembrane unstable protein,which belongs to the F-box-5 protein conserved domain and has a high homology with Erythranthe guttatus COI1.The ORF of the patchouli JAR1 gene was 1725 bp,which encoded 575 amino acids.JAR1protein mainly located in the nucleus,and was a non-secretory,non-transmembrane stable protein,which belongs to the GH3 superfamily and has a high homology with Sesamum indicum JAR1.3.Through heterologous expression in escherichia coli,the optimal conditions to express MYC2-Trx A fusion protein was 0.25 mmol·L^-1 IPTG,induced at 20℃ for 10 h,and a large amount of soluble MYC2 protein was obtained.4.Constructed plant expression vectors of patchouli MYC2 gene overexpression（GBD）and antisense RNA silencing（GR）successfully.Three MYC2 overexpression plants and two antisense RNA silencing plants of Pogostemon cablin were identified.5.DXS,DXR,MCT,CMK,MCS,HDS,HDR genes of Patchouli MEP pathway were the highest expression in leaves and AACT,HMGS,HMGR,MVK,PMK,MVD genes of Patchouli MVA pathway were the highest expression in roots.