Regulation Mechanism Analysis Of SmCPS1 From Salvia Miltiorrhiza Based On Polymorphism And Methylation Pattern Of The Promoter

Gene of copalyl diphosphate synthase 1 of Salvia miltiorrhiza Bge.(SmCPS1)encodes the first key enzyme in the downstream pathway of tanshinones metabolism.Its expression is of great importance to the accumulation of tanshinones contents.Promoter is one of the key factors deciding the spatio-temporal expression of a gene.Sequence variation and methylation changes on a promoter both may change the expression pattern of the gene.By investigating 1)the SmCPS1 gene promoter polymorphism from different genotypes and 2)methylation pattern of SmCPS1 gene promoter in root,stem and leaf tissues,this study tries to explore the regulation mechanism of SmCPS1 gene.The main result are as follows:1)Promoter region of SmCPS1 gene is richer in polymorphic sites than coding regions and some of these sites locates in the key regulatory region of SmCPS1 gene,such as:MIKC_MADS,C2H2,ARR-B,CPP,MYB,AP2,MIKC-MAC,TCP,Dof and TATA-box.These transcription factors are involved in formation of organs like flower and seed,light regulation of genes of carbon metabolism pathway,and response to GA,auxin,salt stress,cold stress and drought stress.2)In different genotypes,the expressions of SmCPS1 gene are not the same;Mantel's test shows SmCPS1 gene expression is in significantly relationship only with dihydrotanshinone I;Pearson correlation analysis shows SmCPS1 gene expression is in significantly relationship only with cryptotanshinone.3)The correlation coefficient between SmCPS1 gene sequence polymorphism and tanshinones contents were slightly improved when only functional polymorphic loci were considered.4)Mantel's test shows that SmCPS1 gene sequence polymorphism showed a biggest value of correlation coefficient with cryptotanshinone,then tanshinone IIA and 1,2-dihydrotanshione.5)The methylated cytosines of SmCPS1 gene promoter region are mainly distribute on the region between-750 bp to-500 bp upstream the translation start site;Transcription binding sites in this region are: NAC,Trihelix,MYB,G2-like,BBR/BPC,Dof,AP2,TCP and MIKC_MADS.6)Twenty one tissue-differential methylated sites were found to be of significant relationship with SmCPS1 gene expression.Some of these sites locates in transription factor binding sites such as: AP2,NAC,BBR/BPC,MIKC_MADS,ERF and C2H2;These transcriotion factors are invloved in responses to pathogen stimuli and environmental stress,regulation of plant such as aging,cell differentiation,.xylem formation.From the aspect of promoter polymorphism and characteristics of promoter methylation from different tissues,this study provide the basis for the regulation mechanism of SmCPS1 gene.This study laid the foundation for further development of SmCPS1 gene promoter sequence as DNA markers of S.miltiorrhiza,as well as providing evidence for the involvement of DNA methylation in the regulation of gene expression.