The Function Of Five SmGRAS Transcription Factors In Regulating The Biosynthesis Of Tanshinones And Phenolic Acids In Salvia Miltiorrhiza

As one of the important traditional Chinese medicines,Danshen(Salvia miltiorrhiza Bunge)has an increasing market demand year by year,and is also a "model medicinal plant".The content of tanshinones and phenolic acids in its dried roots is an important index to evaluate the quality of medicinal materials.Therefore,it is of great significance to study the metabolic regulation mechanism of its active components.GA plays an important role in the regulation of plant growth and development.Although studies have shown that GA treatment can significantly increase the content of tanshinones and phenolic acids,its regulatory mechanism is not clear.As a key regulator of the GA signaling pathway,plant-specific GRAS protein family has been reported to participate in the regulation of GA signaling pathway,root growth and development,stress tolerance and many other biological processes,but its role in the regulation of secondary metabolism is still unclear.Therefore,in this study,the hairy roots of S.miltiorrhiza was used as the material to analyze the regulatory effects of five SmGRAS genes on the growth of hairy roots and the biosynthesis process of tanshinones,phenolic acids and GA,and the regulatory mechanism of promoting the biosynthesis of tanshinones was further analyzed.The following main results are obtained:1.After GA treatment of wild-type hairy roots of S.miltiorrhiza,the dry and fresh weight,tanshinones and phenolic acids contents of hairy roots were significantly increased.Transcriptome analysis of GA treated hairy roots and control.It found the GA mainly control the secondary metabolism process,Mapman's further analysis of genes with secondary metabolic pathway differences shows that most of the differentially expressed genes(DEGs)in secondary metabolism biosynthesis pathway are induced by GA,especially the shikimic acid pathway,MVA pathway,phenols,betaine,wax,and anthocyanins.DEGs in the GA signaling pathway were also analyzed,and it was found that the expressions of GA receptor GID1 and most of GRAS families genes were significantly increased.2.Thirty-five SmGRASs family genes were identified in genome-wide and bioinformatics analysis was carried out.Phylogenetic tree analysis showed that the SmGRASs family was divided into 10 subfamilies.Amino acid sequence alignment showed that all 35 SmGRASs proteins had conserved GRAS domains,but SmGRASs proteins of different subfamilies had different motifs,so they might play different functions.The structural composition and physicochemical properties of 35 SmGRASs genes were further analyzed.In addition,the transcriptome data screening results showed that the transcription level of most SmGRASs was significantly affected by GA.3.Five SmGRAS genes with a significant response to GA in the hairy roots of S.miltiorrhiza were screened and cloned,and the tissue-specific expression patterns of SmGRAS1~5 were analyzed.It was found that all of them had the highest expression in the root periderm and some different expressions in other parts.Subcellular localization of tobacco protoplasts suggests that SmGRAS1~5 proteins are located in the nucleus,and may function as transcription factors.Transcriptional self-activation experiments showed that SmGRAS1/3 had transcriptional self-activation activity,while SmGRAS2/4/5 did not.Analysis of promoter elements in SmGRAS1~5 showed that they all contained GA response elements,as well as other hormones(ABA,MeJA and Aux),light response and abiotic stress response elements.Under the treatment of GA,MeJA,ABA,NAA and SA,the expression levels of most SmGRAS1~5 varied significantly,but there were differences in time and trend,indicating that SmGRAS1~5 had different functions in the hormone signaling pathway.4.The SmGRAS1~5 overexpression and antisense expression hairy roots were obtained respectively.It was found that the growth of the SmGRAS1~5 overexpression hairy roots was inhibited,and the dry and fresh weight were significantly reduced.Overexpression of SmGRAS1~5 can significantly increase the content of tanshinones and decrease the content of GA.However,the regulation of phenolic acids was different.Overexpression of SmGRAS1/2/3/5 reduced the phenolic acids content,while the overexpression of SmGRAS4 increased the phenolic acids content.In SmGRAS1~5 antisense expression lines,the variation trend of components was reversed to the overexpression lines.Analysis of the expression of tanshinone,GA and phenolic acid biosynthesis pathway genes showed that most of the changes in gene expression were consistent with the results of composition content.Among them,the synthase gene SmKSL1 in the downstream of the tanshinones biosynthesis pathway was the most significant change,followed by SmCPS1.5.Yeast one hybridization results showed that SmGRAS1/3/4/5 could bind to the SmKSL1 promoter region,and the EMSA experiment further demonstrated that SmGRAS1/3/4/5 could bind to the GARE-motif elements in the SmKSL1 promoter region.Dual-LUC experiments showed that SmGRAS1/3/4/5 could activate the expression of SmKSL1 promoter,indicating that SmGRAS1/3/4/5 could promote the biosynthesis of tanshinones by directly activating the expression of SmKSL1,which is the key synthase gene in the downstream of the tanshinones biosynthesis pathway.This study found the role of SmGRAS1~5 in regulating the growth of hairy roots,the biosynthesis of tanshinones,GA and phenolic acids,and analyzed their bioinformatics,expression patterns,induced expression and target gene interactions.