Transcription Factor SmWRKY20 Regulating The Biosynthesis Of Effective Components Of Salvia Miltiorrhiza

Salviae Miltiorrhizae Radix et Rhizoma,usually called danshen in China,as a traditional Chinese medicine inherited for thousands of years.It has the effects of stimulating blood circulation and smoothing away congestion,inducing menstruation to relieve pain,which has been recognized by men of traditional Chinese medicine of all dynasties.Modern pharmacological studies have found that danshen plays an important role in the treatment of cardiovascular and cerebrovascular diseases,and can also assist in anti-tumor and liver fibrosis.It has become a pioneer of traditional Chinese medicine toward the ward.Synthetic biology has made great progress in the synthesis of active components of traditional Chinese medicine.In recent years,with the development of genomics,transcriptomics,metabonomics and proteomics in medicinal plants,the functions of enzymes in the biosynthesis pathway of tanshinone and salvianolic acid,the medicinal active components of Salvia miltiorrhiza,have been continuously identified,especially the analysis of tanshinone biosynthesis pathway has made a great breakthrough.Transcription factors are the most basic and important regulation of signal transduction pathways in plants.Among them,WRKY,AP2/ERF,MYB and b HLH play an important role in the regulation network of plant secondary metabolism biosynthesis pathway.WRKY transcription factor is one of the largest transcription factor families in plants.It is named because it contains a conserved WRKYGQK motif.It can recognize the specific nucleotide sequence W-box（T/TGACCA/T）in the promoter of the target gene and participate in plant growth,development,response to biological stress,abiotic stress and plant secondary metabolism biosynthesis.The results showed that WRKY transcription factor was involved in the regulation of biosynthesis of artemisinin in Artemisia annua,vinblastine in Catharanthus roseus and tanshinone in Salvia miltiorrhiza.In this study,based on the latest research results of the laboratory-the genome data of Salvia miltiorrhiza combined with the transcriptome data of hairy roots of Salvia miltiorrhiza induced by yeast extract（Ye）and silver ion（Ag⁺）,the key gene bait of tanshinone biosynthesis pathway and salvianolic acid biosynthesis pathway were used for co expression analysis.The results suggest that the WRKY transcription factor SMILT032209 may be involved in the regulation of tanshinone and salvianolic acid biosynthesis.The open reading frame（ORF）sequence of this gene was obtained by gene cloning technology.Blast in NCBI database showed that the gene had high homology with the registered transcription factor Sm WRKY20 of Salvia miltiorrhiza（99.49%）,so the gene was named Sm WRKY20.Firstly,the bioinformatics analysis of the gene showed that the ORF of the gene was 987 bp,encoded 328 amino acids and had a molecular weight of 35.59 k Da.It had a conserved domain composed of C₂H₂(C-X₅-CX₂₃-HXH)zinc finger structure and WRKYGQK conserved motif,belonging to WRKYⅡd subfamily;The result of spatiotemporal expression analysis,the expression of Sm WRKY20 in hairy roots of Salvia miltiorrhiza increased sharply at 36h by plant hormone,abscisic acid（ABA）.It indicated that exogenous hormones ABA can induce the expression of Sm WRKY20;Sm WRKY20 is mainly expressed in roots and has significant tissue specificity,which is 4 times that of leaves;The Sm WRKY20-GFP fusion expression vector was constructed and transiently expressed in tobacco leaves mediated by Agrobacterium GV3101.By observing the location of Sm WRKY20 fusion green fluorescent protein（GFP）,the results showed that Sm WRKY20 was located in the nucleus,which was consistent with the localization characteristics of most transcription factors.The prokaryotic expression vector p MAL-c2X-Sm WRKY20 was constructed and transformed into the expression competent state of E.coli BL21.The target protein,MBP-Sm WRKY20,was successfully expressed after inducted by IPTG.The overexpression vector p K7WG2D-Sm WRKY20 and RNAi interference expression vector p K7GWIWG2D-Sm WRKY20 were constructed by Gateway technology,and the sterile explants of Salvia miltiorrhiza were infected by Agrobacterium C58C1,obtained the overexpression lines WRKY20 and RNAi interference expression lines wrky20-RNAi.Determination of the content of the salvianolic acid and salvianolic acid B in the transgenic hair roots by HPLC.Comparing with the wild types,for the content of salvianolic acid,the overexpression lines WRKY20 were increased by 16-40%,then the RNAi interference expression lines wrky20 were decreased by 8-37%.And the content of salvianolic acid B was increased by 13-40%in the overexpression lines than the wild types,contrarily the RNAi interference expression lines was decreased by 15-40%.Trough the yeast self-activation and yeast single hybrid experiment,it was found that Sm WRKY20 doesn’t have transcriptional activation activity,but could combine with W-box in the promoters of most key enzyme genes of the biosynthesis pathway of salvianolic acid,including Sm PAL1,Sm C4H1,Sm4CL2,Sm TAT1 and Sm RAS1.It indicated that transcription factor Sm WRKY20,a positive regulatory factor,may directly combine with the promoters of these genes to promote the accumulation of salvianolic acid biosynthesis.In conclusion,existing results are preliminarily speculated that Sm WRKY20 may be involved in the regulation of salvianolic acid synthesis and accumulation through Sm PAL1,Sm C4H1,Sm4CL2,Sm TAT1 and Sm RAS1 genes,which lays a foundation for clarifying the molecular mechanism of salvianolic acid biosynthesis.