Molecular Mechanism Of BZIP Transcription Factors In Regulating The Biosynthesis Of Bioactive Ingredients In Salvia Miltiorrhiza

Objective: Salvia miltiorrhiza is a traditional Chinese medicinal herb and its active components,tanshinones and salvianolic acids,are commonly used in clinical medicine for the treatment of cardiovascular and cerebrovascular diseases.Transcription factors(TFs)are a class of regulators that are widely involved in plant life processes.Previous studies reported that TFs such as MYB,b ZIP,WRKY,NAC,b HLH,might participate in plant secondary metabolism and b ZIP TFs play important roles in hormone signal transduction pathways of plants.In this study,we explored the b ZIP TFs that might regulate the biosynthesis of active components in S.miltiorrhiza with the aim to elucidate the biosynthesis and regulation of active ingredients in S.miltiorrhiza.Methods: The differentially expressed genes were obtained based on comparative transcriptomic analysis of the S.miltiorrhiza hairy root and the b ZIP TFs of A,D and G subgroups were selected according to literatures.We selected these b ZIP TFs that might be related to the regulation of secondary metabolism in S.miltiorrhiza.Then we analyzed the correlation between these TFs and the key pathway genes in the active ingredients biosynthesis pathway by co-expressed assays.Cis-elements and TFs binding sites in the promoter regions of tanshinones and salvianolic acids biosynthetic genes were predicted and yeast one-hybrid(Y1H)and electrophoretic mobility shift assay(EMSA)were used to verify the binding between the b ZIP TFs and the predicted cis-elements.Furthermore,regulators possibly mediate those b ZIP TFs were explored to analyze the possible molecular mechanism of b ZIP-mediated regulation of the biosynthesis of bioactive ingredients in S.miltiorrhiza.Results:1.The trends of transcript levels of different b ZIP TFs were different under ABA treatment,among which,the transcript levels of b ZIP3,b ZIP37 and b ZIP39 increased,the transcript levels of b ZIP8,b ZIP9,b ZIP33,b ZIP71 and b ZIP74 decreased,the transcript levels of b ZIP10,b ZIP18,b ZIP19,b ZIP24,b ZIP36,b ZIP38,b ZIP62,b ZIP68 and b ZIP73 first increased and then decreased,and the transcript levels of b ZIP67 first decreased and then increased.2.After ABA treatment,the expression of b ZIP3,b ZIP6,b ZIP10,b ZIP18,b ZIP19,b ZIP30,b ZIP36,b ZIP37,b ZIP38,b ZIP39,b ZIP68,b ZIP71 was correlated with the expression of active ingredients biosynthesis pathway genes.The results of correlation analysis showed that: b ZIP3 was positively correlated with CYP76AK1,b ZIP6 and b ZIP10 were positively correlated with CYP76AH1,b ZIP18 was positively correlated with CYP76AH1,b ZIP19 and b ZIP37 were positively correlated with CYP76AK1,b ZIP30 was positively correlated with HPPR,b ZIP36 was positively correlated with CYP76AH3,b ZIP38 was positively correlated with TAT,b ZIP39 was positively correlated with CYP76AK1 and 4CL2,b ZIP68 was positively correlated with CYP76AH3,b ZIP69 was negatively correlated with TAT,b ZIP71 was significantly negatively correlated with CYP76AK1.3.Cis-elements in the promoter regions of 30 pathway genes were predicted,the ABRE elements(ABA-responsive element)were located in the promoter regions of 24 pathway genes,which was reported that b ZIP TFs could bind to ABRE elements to regulate the expression of genes).At the same time,the promoter sequences of 30 pathway genes were analyzed to further predict the TFs binding sites in Plant TFDB,it was predicted that there were 26 pathway gene promoters could bind to b ZIP TFs.4.Partial b ZIP TFs and five pathway gene promoters regions have been cloned.Then Y1 H was performed according to the predicted binding relationships.The results showed that b ZIP6 could bind to the fragment of 4CL2 promoter,and b ZIP18 and b ZIP19 could bind to 4CL2 and TAT promoter fragments.Results of EMSA verified that b ZIP6,b ZIP18 and b ZIP19 were bound to the ABRE element in the promoters.5.b ZIP TFs could be phosphorylated as the substrates of Sn RK2 in the ABA signal transduction pathway.We got 10 unigenes annotated as Sn RK2 in the danshen transcriptome,and three of them belonged to Ⅲ subclass by clustering analysis.We speculate that these Sn RK2 s could activate the b ZIP TFs to regulate the transcription of downstream biosynthetic pathway genes.Conclusion: In this study,we analyzed the danshen transcriptome and found 13 b ZIP TFs in S.miltiorrhiza that might be involved in tanshinones and salvianolic acids biosynthesis.Among them,b ZIP6 could bind to ABRE element in 4CL2 promoter,b ZIP18 and b ZIP19 could bind to ABRE element in 4CL2 and TAT promoter.These results indicated that the b ZIP TFs could regulate the expression of pathway genes by binding to the promoter regions to modulate the biosynthesis of active components in S.miltiorrhiza.What’s more,the transcript levels of b ZIP6,b ZIP18 and b ZIP19 were all induced by ABA.Thus,it was speculated that these b ZIPs might regulate the expression of biosynthetic pathway genes through ABA signalling pathway to regulate the biosynthesis of the active components in S.miltiorrhiza.