The Functional Study Of SmWRKY9 Transcription Factor In Salvia Miltiorrhiza

Salvia miltiorrhiza Bunge(S.miltiorrhiza),a dicotyledonous herb of the Lamiaceae family and the genus Salvia,is one of the traditional Chinese herbal medicines,mainly used for the treatment of cardiovascular and cerebrovascular diseases.Cardiovascular and cerebrovascular diseases is the number one killer of human death due to the high incidence rate.This lead to a very huge demand of S.miltiorrhiza.However,as the yield and quality of artificially-planted S.miltiorrhiza decline,the demand of S.miltiorrhiza exceeds the supply.It is one of the effective ways to use biotechnology like genetic engineering to increase the yield of active ingredients in S.miltiorrhiza.WRKY transcription factors are one of the largest transcription factor families,which can be widely found in plant.It is named for its special conserved domain WRKYGQK.WRKY transcription factors are often involved in the plant stress response.Recently,it has been reported that WRKYs are also involved in the regulation of the secondary metabolites synthesis.In this study,we firstly analyze the transcriptomic database sequenced from MJ induced hairy roots of S.miltiorrhiza,and obtained a MJ-responsive WRKY gene.The cDNA sequence was cloned by PCR and was used to perform Blast on NCBI.Based on the high homology(98%)with reported SmWRKY9,it was named SmWRKY9.Tissue expression profile showed the expression of SmWRKY9 was highest in the main root of S.miltiorrhiza.It was observably induced by MJ and YE based on the results of hormone-induced expression profile.The expression level increased quickly after MJ induction and reached the highest point in 1h,which was 7 times as many as the control;its expression level reached the highest point in 2h after YE induction,which was 5.6times as many as the control.In addition,the expression level of SmWRKY9 was slightly up-regulated by the induction of ET and SA and slightly down-regulated by the induction of ABA.Based on subcellular localization experiments,SmWRKY9 was located in the nucleus.Transgenic hairy roots of SmWRKY9 overexpression and SmWRKY9-SRDX were obtained by genetic transformation using sterile S.miltiorrhiza explants mediated by C58C1.The expression level of key enzyme genes in the rosmarinic acid biosynthesis pathway was detected by qRT-PCR in SmWRKY9 transgenic hairy roots.The results showed that overexpression of SmWRKY9 could up-regulate SmRAS1,SmCYP98A14 and Sm4CL1.At the same time,the expression of SmRAS1 and SmCYP98A14 in SmWRKY9-SRDX transgenic hairy roots decreased significantly.The content of rosmarinic acid was determined by HPLC.The results showed that the content of rosmarinic acid in SmWRKY9 overexpression lines was higher than that in the control lines(9.8373 mg/g DW),and the highest content of rosmarinic acid in SmWRKY9-34 lines was reached to 21.83182 mg/g DW.In contrast,the SmWRKY9-SRDX lines had a lower rosmarinic acid content than the control lines,and the SmWRKY9-SRDX-24 lines had a minimum rosmarinic acid content(3.76067 mg/g DW).Salvianolic acid B decreased in both SmWRKY9 overexpression and SmWRKY9-SRDX lines,and the lowest content of salvianolic acid B in SmWRKY9-SRDX-24 lines was 0.48 mg/g DW.Dual luciferase reporter gene assays showed that SmWRKY9 played a positive regulatory role in the expression of SmRAS1 and SmCYP98A14.Yeast single-hybrid experiments and EMSA assays showed that SmWRKY9 interacts with the W-box in the SmRAS1 and SmCYP98A14 promoter.In addition,we also performed RNA-seq analysis using SmWRKY9 overexpression hairy root SmWRKY9-26,and found that overexpression of SmWRKY9 also increased the expression levels of SmPAL3,Sm4CL2,Sm4CL3,and Sm4CL8,suggesting that they may participate in the SmWRKY9-mediated pathway.In summary,SmWRKY9 can promote the biosynthesis of rosmarinic acid by positively regulating the expression of SmRAS1 and SmCYP98A14.