| Contents of phenolic acids and tanshinones in Salvia miltiorrhiza Bunge are the key quality indicator to evaluate Chinese herb medicine,Danshen.It is very important to illuminate the regulation mechanism of phenolic acids and tanshinones biosynthesis which could help us got high quality Danshen.Biosynthesis of phenolic acids and tanshinones are affected by both environment and genetic.Transcription factors(TFs)could regulate genes' expression directly as well as take part in the regulation of environment factors on plant secondary metabolism.Reveal the regulation mechanism of TFs on S.miltiorrhiza secondary metabolism is the premise of S.miltiorrhiza metabolism engineering research and can also lay a foundation for elucidation of the quality formation mechanism of S.miltiorrhiza.In this study,the molecular mechanism of 5 bHLH family TFs of S.miltiorrhiza on regulating phenolic acids and tanshinones was studied,and the synergistic regulation mechanism of SmbHLH7 and SmMYB39 on phenolic acids was described.The specific results are as follows:1.Five complete TFs gene of the bHLH family were screened from the S.miltiorrhiza transcriptome database,which were named as SmbHLH3,SmbHLH7,SmbHLH148,SmbHLH10 and SmbHLH130,respectively.Typical HLH domain were found in these five TFs by comparative analysis with other databases.All these five bHLHs present in the six parts(stem,leaf,flower,root epidermis,xylem and fibrous root)of two years old flowering S.miltiorrhiza,but with different expression patterns.The five bHLHs expression were also being induced by MeJA,ABA and GA.Results of subcellular localization showed that GFP fluorescence of these proteins all present in nucleus,and fluorescence of SmbHLH10-GFP also present in other places in the cell with lower luminance.2.The gene overexpression hairy roots of five SmbHLHs were obtained,respectively.We found that overexpression of SmbHLH3 inhibited the accumulation of both tanshinones and phenolic acids in S.miltiorrhiza hairy roots.Phenolic acids and tanshinones biosynthesis were induced in transgenic hairy roots of SmbHLH7,SmbHLH148 and SmbHLH130,respectively.Overexpression of SmbHLH10 improved tanshinones accumulation while showed non-significant effect on phenolic acids biosynthesis.The main enzyme genes' expression of phenolic acids and tanshinones biosynthetic pathways were changed significantly in transgenic hairy roots when compared to WT.Results of yeast one-hybrid assay showed that SmbHLH3 could binds the predicted G-box motifs within the promoters of TAT and HPPR,while SmbHLH7,SmbHLH148,SmbHLH130 could binds the promoters of C4H1,TAT,HPPR,CYP98A14.Moreover,SmbHLH148 could also binds the G-box motifs within the promoters of PAL1.For tanshinones biosynthesis pathway,SmbHLH3 could interact with promoters of KSL1 and CYP76AH1,SmbHLH7,SmbHLH148 and SmbHLH130 could interact with promoters of DXS2,CPS1,CYP76AH1.SmbHLH7 and SmbHLH130 could also interact with G-box within KSL1 promoter.The interactions of SmbHLH130 with KSL1 or CYP76AH1 were weaker than others.SmbHLH10 could binds the predicted G-box motifs within the promoters of DXS2,CPS1 and CPS5.3.Previously we found that SmMYB39 was a R2R3 MYB which regulated phenolic acids biosynthesis negatively.Considering that bHLH transcription factors generally interact with MYB protein to form a complex to regulate secondary metabolism,we screened these 5 SmbHLHs whether it could interact with SmMYB39 by yeast two-hybrid assay.The results showed that SmbHLH7 and SmbHLH130 could both interact with SmMYB39.The interaction between SmbHLH130 and SmMYB39 was weakly.The sequence analysis of SmbHLH7 and SmMYB39 proteins showed that the interaction region of SmbHLH7 and SmMYB39 protein may located at the N terminal of each other,which was confirmed by yeast two-hybrid experiment after the protein was truncated.4.We got the overexpression and RNAi silencing hairy roots of SmMYB39.Found that SmMYB39 suppressed tanshinones biosynthesis as well as phenolic acids.We also got RNAi silencing hairy roots of SmbHLH7,in which phenolic acids and tanshinones contents were decreased when compared with WT.Analyzed of genes expression of phenolic acids and tanshinones metabolic pathways in transgenic hairy roots revealed that SmMYB39 and SmbHLH7 could regulate the same genes.The co-overexpression of SmbHLH7 and SmMYB39 showed that the expression level of SmMYB39 was increased to several hundred times of the control group,while the expression level of SmbHLH7 was only increased by less than 10 times.The biological synthesis of phenolic acids was inhibited,while the content of tanshinone was increased.When SmbHLH7 was overexpressed and SmMYB39 was silenced at the same time,the accumulation of phenolic acids and tanshinones were also promoted,and the promotion ratio compared to WT were higher than that of the hairy root of SmbHLH7 overexpression or SmMYB39 RNAi alone.The transcription of SmbHLH7 was inhibited in the overexpressed hairy roots of SmMYB39,while it was increased after the silencing of SmMYB39.Conversely,overexpression of SmbHLH7 promotes transcription of SmMYB39,which was inhibited by silencing SmbHLH7.These results indicated that in addition to the interaction between SmMYB39 and SmbHLH7 at proteins level,they could co-adjust at transcription level.In the present study,we got five bHLHs from S.miltiorrhiza transcriptome database which could regulate phenolic acids or tanshinones biosynthesis.Then,we analyzed their function by over expression in hairy roots and identified their target genes which involved in phenolic acids and tanshinones biosynthesis pathways.In addition,we found the synergistic regulatory mechanism of SmbHLH7 and SmMYB39 had three levels: SmMYB39 can competitively bind key genes of phenolic acids or tanshinones metabolic pathway with SmbHLH7,negative regulator SmMYB39 directly inhibits SmbHLH7 transcription,SmMYB39 and other MYB competitively interact with SmbHLH7,preventing the formation of the positive-regulating MBW complex. |
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