Molecular Mechanism Of SmVQ16-SmWRKY44 Module Regulation Of Tanshinone Biosynthesis In Salvia Miltiorrhiza

Salvia miltiorrhiza,as a large traditional Chinese medicine,has important economic and medicinal value,and the market demand is enormous.Tanshinone is a diterpene quinone compounds,which is not only its main fat-soluble active component,but also a key indicator of the quality of its medicinal materials.The biosynthesis pathway of tanshinone is being gradually decoded.The regulatory network of tanshinone biosynthesis needs to be enriched and improved.The discovery of key regulatory factors can provide candidate targets for improving the quality of salvia miltiorrhiza medicinal materials.We previously identified a WRKY transcription factor Sm WRKY44,a member of IIc subgroup,which directly acts on the upstream of Sm CPS1 and Sm KSL1,activating the transcription of both and promoting tanshinone biosynthesis.In this test,Sm WRKY44 was used as bait to screen c DNA library of Salvia miltiorrhiza through yeast two hybrid,and several VQ proteins were identified.To further study the interaction between Sm WRKY44 and VQ protein,VQ gene family was cloned and identified by combining genome and transcriptome analysis.This study focuses on the molecular mechanism of the Sm VQ16-Sm WRKY44 module regulating tanshinone biosynthesis through activating Sm CPS1/Sm KSL1 transcription.The main results are as follows:1.Eighteen VQ proteins in Salvia miltiorrhiza were cloned and identified.Expression analysis showed that the expression sites of VQ gene were mainly in leaves and roots,and the expression of most VQ genes could respond to exogenous elicitors JA and SA.Y2 H test showed that except Sm VQ1 and Sm VQ10,the other 16 VQ proteins interacted with Sm WRKY44,of which 16 VQ proteins interacted with Sm WRKY44.Sm VQ16 homologous to Arabidopsis MKS1 was selected for the next experiment.2.Using Pull-down and CO-IP tests,it was proved that there was an interaction between Sm WRKY44 and Sm VQ16 at protein level in vivo and in vitro.3.Transgenic hairy roots were obtained by constructing Sm VQ16 overexpression and RNAi vector and using C58C1 mediated genetic transformation.The results showed that overexpression of Sm VQ16 increased the accumulation of tanshinone I,IIA and cryptotanshinone,and the expression of Sm WRKY44 target genes was significantly increased in Sm VQ16 overexpression lines,while the expression of Sm WRKY44 target genes was significantly decreased in RNAi lines.4.EMSA test showed that Sm VQ16 could increased the ability of Sm WRKY44 to bind the promoters of target genes(Sm CPS1,Sm KSL1)in vitro.The dual-Luc report showed that Sm VQ16 increased the ability of Sm WRKY44 to activate the transcriptional activity of promoters of two downstream target genes.5.Using the Cell Free Degradation experimental system,the recombinant protein expressed by Sm WRKY44 in vitro was co-incubated with the total protein of Salvia miltiorrhiza WT plants,Sm VQ16 overexpressed plants and RNAi plants.Using MG132 and WB technology,it was verified that Sm VQ16 increased the stability of Sm WRKY44 by inhibiting the ubiquitination degradation of Sm WRKY44 protein.In conclusion,this study revealed the molecular mechanism of positive regulation of tanshinone biosynthesis by Sm VQ16 interacting with Sm WRKY44,enhancing Sm WRKY44’s ability to promote Sm CPS1 and Sm KSL1 transcription and inhibiting the 26 S proteasome degradation of Sm WRKY44 protein.