| Salvia miltiorrhiza is a kind of perennial erect herb plant,its roots and rhizomes are used in Chinese traditional medicine and has been widely used for the treatment of cardiovascular and cerebrovascular diseases.The liposoluble tanshinones and the hydrosoluble phenolic acids consist of its main medicinal active components.Nowadays,due to the increasing market demand and the low quality of traditional S.miltiorrhiza plants,the shortage of resource in the market has become a serious problem.Thus,the research on biosynthesis pathway and metabolic engineering of tanshinones and phenolic acids has become a hot spot.Jasmonic acid(JA)is a kind of hormone which is widely exist in various plants.It has been proved to be able to induce the accumulation of secondary metabolites in plants and regulated by the upstream transcription factors such as bHLHs.In this study,we firstly identified 131 bHLH genes from our local transcription database.According to the transcriptome database induced by MJ&YE,we successfully selected a bHLH transcription factor which was significantly induced by MJ and named it as SmbHLH7.Then we introduced them into the pHB vector.Meanwhile,the C58C1 strain contained the recombinant plasmid was used to infect the aseptic explants to acquire the transgenic hairy root lines and the empty pHB was regarded as the control.Then,a series of biochemical experiments were performed to further analyze the function of SmbHLH7.The results has been presented as follows:(1)Bioinformatics analysis: the results showed that SmbHLH7 has high homology with MYC2 in Sesamum indicum and the multiple sequence alignment analysis indicated that SmbHLH7 had a conservative MYC domain and a HLH domain.We also noticed that SmbHLH7 has similar expression model with the key enzyme genes of tanshinone pathway,SmDXS2,SmAACT,SmCPS1 after the MJ treatment based the data of our local transcriptome database.(2)Identification of SmbHLH7: We used the leaves of S.miltiorrhiza as template and successfully obtained a full-length ORF sequence which is 1290 bp andcodes 429 amino acids.(3)Tissue expression profiles: the results showed that SmbHLH7 has low expression in taproot,fibrous roots and petioles,but particularly expresses in leaves.(4)Induction expression profiles: we noticed that the expression level of SmbHLH7 significantly rose after exogenous hormone treatment,especially for MeJA:arising to about 80-fold at 2h.(5)Acquisition of SmbHLH7 transgenic hairy roots: we constructed the overexpression vector pHB-SmbHLH7-YFP and acquired 10 positive lines while 11 empty vector transgenic lines were obtained.(6)Expression of tanshinones and phenolic acids biosynthetic genes in SmbHLH7 transgenic hairy roots: The results of qRT-PCR analysis showed that:overexpression of SmbHLH7 can significantly up-regulate SmHMGS?SmGGPPS1?SmCYP76AH1?SmKSL1 while down-regulate SmRAS1,SmCYP98A14,SmTAT1.(7)The contents of tanshinones and phenolic acids in SmbHLH7 transgenic hairy roots lines: we found that the transcription factor can promote the accumulation of tanshinones in S.miltiorrhiza hairy roots(Among them,SmbHLH7-OE-15 line accumulated the highest content of total tanshinone to 8.89mg/g which was 4.1 times of the control)but repress the production of phenolic acids.(8)Yeast one-hybrid Assays showed that SmbHLH7 can binding to the promoter of SmRAS1.(9)Dual-luciferase assays: the result indicated that SmbHLH7 can bind to the promoter of SmRAS1 and SmTAT1 and repress the expression of them and implied that they may the direct regulation targets of SmbHLH7.(10)Yeast two-hybrid and BiFC assays: we found SmbHLH7 can interact with SmJAZ3 L through Yeast two-hybrid and BiFC assays.In general,we successfully cloned a bHLH transcription factor SmbHLH7.Thereafter,the regulate function of SmbHLH7 in tanshinones,phenolic acids biosynthesis pathways was further analyzed,and its regulation mechanism was also preliminarily revealed.All these results may provide a better understanding of the synthesis and regulation mechanism of tanshinones and phenolic acids and alsoprovides a certain experimental basis for the analysis of the regulation pathway of the biosynthesis pathway of tanshinones and phenolic acids... |
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