Overexpression Of SmbHLH53 Increases The Accumulation Of Salvianolic Acid

Salvia miltiorrhiza Bunge,a perennial medicinal herb,which medicinal parts is roots and rhizomes.The active ingredients in S.miltiorrhiza are tanshinones and phenolic acids,which is used to treat various diseases such as coronary heart disease,cerebrovascular disease,neurodegenerative diseases,insomnia and bone loss,etc.Increasing the content of phenolic acids or tanshinones in S.miltiorrhiza by plant genetic engineering is the current research hotspot.It is known that jasmonic acid and its derivatives can effectively increase the content of secondary metabolites in S.miltiorrhiza.The JAZ,MYC2 and other proteins on the JA signaling pathway are involved in the regulation of secondary metabolite biosynthesis.The JAM(JASMONATE-ASSOCIATED MYC2-LIKE)protein belongs to the Ⅲd subfamily of the bHLH transcription factor family and is a negative regulator of the response to methyl jasmonate.It is known that,in Arabidopsis thaliana,bHLH transcription factor Ⅲd subfamily members JAM1,JAM2,JAM3 and Ⅲe subfamily members MYC2,MYC3,MYC4 antagonize the downstream biological responses regulated by jasmonic acid.In Taxus cuspidata,the JAM proteins TcJAMYC1,TcJAMYC2,and TcJAMYC4 negatively regulate taxol biosynthetic genes.There is no report on JAM protein in S.miltiorrhiza.In this experiment,the bHLH transcription factor Ⅲd subfamily gene SmbHLH53 was cloned from S.miltiorrhiza,and its secondary metabolism control function was preliminary studied.The main contents and results of this study are as follows:1.Based on the established transcriptome and genome database of S.miltiorrhiza,a bHLH transcription factor gene SmbHLH53 was cloned by PCR.The full length of SmbHLH53 gene is 1799 bp,including an 11bp intron,and its cDNA length is 1788bp,encoding 595 amino acid residues.Phylogenetic analysis indicated that it belongs to theⅢd subfamily and has the highest similarity to JAM1(bHLH17)in A.thaliana.PLANTCARE analysis showed that its promoter region contains light responsive elements,stress response elements,hormone response elements,and protein binding elements.WOLFPSORT predictions show that SmbHLH53 is mainly located in the nucleus and secondarily in the plasma membrane.Real-time PCR results showed that the expression of SmbHLH53 gene was highest in leaves and roots,and SmbHLH53 was a rapid response gene of exogenous ABA and mechanical injury.At the same time,the expression level of SmbHLH53 was significantly up-regulated in the SmMYC2 overexpression strains.2.The subcellular localization expression vector,yeast two-hybrid expression vector and bimolecular fluorescence complementation(BiFC)expression vector of SmbHLH53 were constructed using GATEWAY technology.The subcellular localization of SmbHLH53 showed that the protein was mainly located in the nucleus and also distributed in the plasma membrane.Yeast two-hybrid and BiFC experimental results showed that SmbHLH53 protein can form homodimer,and SmbHLH53 protein can interact with SmJAZ1,SmJAZ3,SmJAZ8.3.Overexpression vector of SmbHLH53 was constructed by using GATEWAY technology.SmbHLH53-overexpression positive lines of S.miltiorrhiza was obtained through the method of Agrobacterium tumefaciens-mediated transformation of leaf discs.Five strains with significant differences were detected by molecular level detection:OE-1,OE-2,OE-3,OE-4,OE-6.Among them,the changes of strains OE-1 and OE-6 were the most obvious,and the expression levels of SmbHLH53 were about 12-fold and 4-fold higher than those of the control group,respectively.4.The results of the determination of the overexpression strains OE-1,OE-6 total phenolic acids and total flavonoids showed that the total phenolic acid content of OE-1 and OE-6 increased significantly,and were 1.29-fold and 1.28-fold of those in the control group,respectively;the total flavonoid content was 2.02-and 1.14-fold of those in the control group,respectively.The changes of secondary metabolites salvianolic acid B,rosmarinic acid,tanshinone IIA,and cryptotanshinone were determined by LC-MS experiments.The contents of rosmarinic acid in OE-1 and OE-6 were 1.15-and 1.41-fold of those in the control group,respectively.The OE-1 and OE-6 levels of salvianolic acid B were 1.08-and 1.39-fold of those in the control group.There was no significant change in the contents of tanshinone IIA and cryptotanshinone.5.Transcriptome sequencing of transgenic strains OE-6 and control strains revealed a total of 3420 differentially expressed genes(DEGs)in OE-6 strains,of which 1672 genes were up-regulated and the remaining 1748 genes were down-regulated.Two genes with significant up-regulated expression were found on the pathway of phenolic acids synthesis:SmRAS5 and Sm4CL9.Compared with the control line,SmRAS5 was up-regulated about 1.1-fold,and Sm4CL9 was up-regulated about 2.3-fold.Most other genes expressed only a slight change.There were three differentially expressed genes in the tanshinone biosynthetic pathway with significant up-regulated expression:SmCPS2,SmGPPS2,and SmGGPPS2,which were up-regulated by 1.34-,4.13-,and 3.08-fold,respectively,compared with the control strains.And one significantly down-regulated gene SmDXS1,compared to the control line,was down-regulated by 2.55-fold.This study preliminarily confirmed the positive regulation effect of S.miltiorrhiza transcription factor SmbHLH53 on the accumulation of salvianolic acid B and rosmarinic acid,and is speculated that its relationship with SmMYC2 in the JA signal pathway of S.miltiorrhiza.It provides a biological basis for enhancing the active ingredients of medicinal plant S.miltiorrhiza.