Interaction Between SmTGA2 And The Promoter Of The Key Enzymes For Salvianolic Acid B Synthesis

Salvia Miltiorrhiza Bunge is a traditional Chinese medicinal material used for the treatment of cardiovascular and cerebrovascular diseases.Salvianolic acid B is the main component of S.miltiorrhiza,which has high medicinal value.The synthesis pathway of rosmarinic acid,a precursor of salvianolic acid B synthesis,has been clearly studied.RAS,PAL and CYP98A14 are the key enzymes in the synthesis pathway of rosmarinic acid.Our previous study found that salicylic acid(SA)as an exogenous inducer could significantly increase the biosynthesis amount of salicylic acid B,but the specific molecular mechanism is still unclear.TGA transcription factors bind to NPR,the receptor protein of SA,and are involved in the plant's antiretroviral response.TGA transcription factors belong to the b ZIP family of plants,which can regulate the transcription of as-1 cis-acting elements under the action of SA.TGAs is found to bind to the SA receptor,NPR1,and plays a crucial role in the SA signaling pathway.Taking SA signaling pathway as the entry point,this study used bioinformatics analysis,Real-Time quantitative PCR(q PCR),Dual-Luciferase Assays(Dual-LUC),Electrophoretic Mobility Shift Assay(EMSA)and other methods to analyze and verify the key enzyme genes related to salvianolic acid B synthesis that may be regulated by TGA2 transcription factor.The following results were obtained:1.SmTGA2 gene of the TGA transcription factor family in S.miltiorrhiza was cloned and identified.After NCBI Blast and Phylogenetic tree analysis,it was identified as the direct homologous gene of At TGA2,so it was named SmTGA2.Quantitative results showed that its expression was regulated by salicylic acid.2.The promoter sequence of SmCYP98A14 was cloned,and promoter analysis showed that the promoter sequences of key enzyme genes SmRAS,SmCYP98A14 and SmPAL1 synthesized by Salvianolic acid B all contained as-1-box,and this cing-acting element was the recognition site of TGA gene family.The quantitative results verified that the key enzyme genes of SmRAS,SmCYP98A14 and SmPAL1 could indeed respond to the induction of salicylic acid.3.Dual-LUC results showed that TGA2 could bind to the promoters of SmRAS,SmCYP98A14 and SmPAL1 and inhibit their expression.4.EMSA results showed that TGA2 protein could bind specifically to as-1-box on SmRAS,SmCYP98A14 and SmPAL1 promoter.In conclusion,this study cloned a TGA transcription factor,TGA2,and screened three key enzyme genes related to salvianolic acid B synthesis regulated by the TGA transcription factor,providing a theoretical basis and experimental basis for the molecular mechanism of SA mediated secondary metabolite synthesis pathway.