Molecular Mechnism Of SmWRKY40 Inhibits The Biosynthesis Of Tanshinone In Salvia Miltiorrhiza

Salvia miltiorrhiza Bunge is a perennial herb of the genus Salvia,which is a commonly used traditional medicinal plant.The active ingredients of salvia miltiorrhiza are mainly tanshinones and salvianolic acids.The content of active ingredients is a key indicator of the quality of medicinal materials.How to increase the content of active ingredients to improve the quality of medicinal materials is the focus of current research.To clarify the key genes of the biosynthetic pathway of active ingredients,and to uncover its transcription regulation can lay a theoretical foundation for regulating the content of active ingredients.Our research team previously used the promoter of Sm CPS1,the key enzyme gene for tanshinone biosynthesis,as a bait,to screening Danshen c DNA library by yeast single-hybrid.A transcription factor Sm WRKY40 was identified.Overexpression of Sm WRKY40 in hairy roots of Salvia miltiorrhiza significantly inhibited tanshinone accumulation.Further,experiments using RT-q PCR,yeast one-hybrid,electrophoretic mobility shift assay,and dual luciferase reporter gene revealed the molecular mechanism that Sm WRKY40 inhibited tanshinone biosynthesis.The main results are as follows:1.The Sm WRKY40 gene was cloned from Salvia miltiorrhiza,with a CDS of 1059 bp and encoding 352 amino acids.Sm WRKY40 was mainly expressed in the roots,the expression level in the stems and leaves was very low.Exogenous Me JA and SA treatment can inhibit transcription of Sm WRKY40.Subcellular localization experiments showed that Sm WRKY40 is localized in the nucleus.2.Transgenic plants and hairy roots of Salvia miltiorrhiza over-expressing Sm WRKY40 were obtained through genetic transformation mediated by A.tumefaciens and Agrobacterium rhizogenes.HPLC test showed that over-expression of Sm WRKY40 resulted in a significant reduction in the content of Tanshinone I and Tanshinone IIA in hairy roots.RT-q PCR was used to analyze the expression level of tanshinone biosynthetic genes.It was found that the expression levels of Sm DXS2,Sm DXR,Sm MDS,Sm HMGR3,Sm GGPPS3,Sm CPS1,and Sm CPS5 were all down-regulated to various degree.The down regulation of Sm CPS1 and Sm CPS5 was significant compared with wild type.It is speculated that these two genes may be downstream target genes of Sm WRKY40.3.To further analyze the molecular mechanism of that Sm WRKY40 inhibiting tanshinone accumulation,yeast one-hybrid was conducted and results showed that Sm WRKY40 can interact with Sm CPS1 and Sm CPS5 promoters in yeast.EMSA confirmed that Sm WRKY40 can directly bind to Sm CPS1 and Sm CPS5 promoters in vitro.Dual fluorescence reporter gene test showed that Sm WRKY40 directly inhibited the transcriptional activity of promoters of Sm CPS1 and Sm CPS5.In summary,this study revealed Sm WRKY40 directly inhibits the transcription of Sm CPS1 and Sm CPS5,thus negatively regulates tanshinone biosynthesis.