Metabolic Engineering With Transcription Factor SmbHLH124 In Salvia Miltiorrhiza

Salvia miltiorrhiza Bunge is one of the most renowned traditional medicinal plant as well as model plant,which belongs to Lamiaceae family.It is primarily used for treating cardiovascular and cerebrovascular diseases.Tanshinones and phenolic acids are the dominated major classes of bioactive components of this plant.Genetic engineering has emerged as an effective strategy for increasing the content of the active components in the plant.SmbHLH124,a member of the b HLH family transcription factors,is highly expressed in root;however,its role in regulation of secondary metabolites of S.miltiorrhiza is not well understood.Genetic transformation of hairy roots was used to analyze the function of SmbHLH124.Overexpression of Smb HLH12 downregulated phenolic acids contents and upregulated tanshinones accumulation,and root biomass.The main research contents and results are as follows:1.The full-length SmbHLH124 was 830 bp in size and consisted of one intron encoding 91 amino acids,which was cloned from S.miltiorrhiza root based on the transcriptome and genome database.2.The SmbHLH124 was transferred into S.miltiorrhiza by Agrobacterium tumefaciens(C58C1)mediated genetic transformation.Three overexpression lines were obtained.The fresh weights in all the transgenic lines were greatly increased.Compared to the control,the fresh weight of SmbHLH124-overexpressing hairy root was increased by 1.46 times.3.The contents of secondary metabolites in transgenic and control roots were determined by high performance liquid chromatography.Compared to the control,the content of rosmarinic acid and salvianolic acid B in over-expressed SmbHLH124 roots were reduced by 1.99 and 3.33 times;the content of dihydrotanshinoneⅠ,cryptotanshinone,tanshinoneⅠand tanshinoneⅡA were increased by 3.69,2.70,2.31 and 1.82 times.4.According to RNA-Seq sequencing analysis,overexpression of SmbHLH124 resulted in the up-regulation of the expression of tanshinone biosynthesis and the downregulation of gibberellin metabolism,whereas salvianolic acid biosynthesis downregulated and lignin biosynthetic genes upregulated.The expression trends of the related enzyme genes in biosynthesis pathway were consistent with the change of the accumulation of metabolites.