The Regulatory Roles Of SmHY5 In The Biosynthesis Of Bioactive Compounds And Lateral Root Development In Salvia Miltiorrhiza

Salvia miltiorrhiza is one of the traditional Chinese medicines,which is widely used for the treatment of a variety of cardiovascular and cerebrovascular diseases.The regulatory mechanisms of biosynthesis of bioactive compounds have been deeply researched,while little is known about the roles of light in accumulation of secondary metabolites in S.miltiorrhia.Meanwhile,as rhizome is the main organ used for medicine in S.miltiorrhiza,the root development and morphology effect the yield and quality of this herb.In this study,we cloned SmHY5,the "core regulator" of light signaling and characterized its function in the accumulation of phenolic acids and tashinones as well as lateral root development in S.miltiorrhiza.The main results are as follows:1.SmHY5 contained an open reading frame of 474 bp encoding 158 amino acids.SmHY5 localized in nucleus and contained a bZIP domain,it was a typical bZIP transcription factor.SmHY5 expressed in root,stem,leaf and flower,with the highest expression in leaf and the lowest expression in root.The expression level of SmHY5 in leaf was 26.74 folds of that in root,this maybe because it manly function after light induction.2.The RNAi and overexpression vector of SmHY5 were constructed.The transgenic plants of S.miltiorrhiza were obtained by GV3101-mediated leaf disc transformation,and the morphological indexes of lateral roots in the transgenic lines were analyzed.The results showed that the number of primary,secondary lateral roots and fresh weight decreased significantly,while the diameter and length of primary lateral roots increased significantly in SmHY5 overexpressed lines.For example,the number of primary lateral roots,secondary lateral roots and root fresh weight in OE-84 were 55.56%,39.47%and 72.50%of those in WT(wild type),respectively,and the diameter and length of primary lateral roots 1.65 and 1.57 fold of those in WT(wild type),respectively.The number of primary lateral roots,secondary lateral roots and root fresh weight increased significantly,while the diameter and length of primary lateral roots decreased significantly in SmHY5 RNAi lines.For example,the number of primary lateral roots,secondary lateral roots and root fresh weight in RNAi-80 were 1.31,1.89 and 1.26 fold higher than those in WT(wild type).respectively,and the diameter and root length of primary lateral roots were 65.79%and 81.71%of those in WT(wild type).respectively.3.The accumulation of phenolic acids in transgenic lines was analyzed.The overexpression of SmHY5 significantly increased the content of phenolic acids in the roots of S.miltiorrhiza.For example,the contents of p-coumaric acid(OE-84),rosmarinic acid(OE-23)and salvianolic acid B(OE-23)were 2.43,1.81 and 1.39 fold higher than those of the wild type control,respectively.The contents of rosmarinic acid and salvianolic acid B decreased significantly,but there was no significant change in p-coumaric acid content in SmHY5 RNAi line.It is worth noting that there was no significant change in the content of salvianolic acid A in SmHY5 overexpression lines.However,the content of salvianolic acid A in SmHY5 RNAi line was significantly higher than that in wild type line.For example,the content of salvianolic acid A in RNAi-18 was 2.67 fold higher than that in wild type line4.Similar to the accumulation of phenolic acids,the content of tanshinone increased significantly in SmHY5 overexpression lines.For example,the contents of cryptotanshinone,tanshinone ?A,tanshinone I and dihydrotanshinone ? in OE-84 were 3.85,6.00,1.83,3.38 times higher than those in WT(wild type),respectively.The content of tanshinone in SmHY5 RNAi lines was decreased significantly.For example,the contents of cryptotanshinone,tanshinone?A,tanshinone ? and dihydrotanshinone I in RNAi-80 were 40.16%,55.56%,75.36%and 13.88%of those in WT(wild type),respectively.5.The RNA-seq was conducted and the differentially expressed genes were analyzed in transgenic lines.6039 differentially expressed genes were screened in SmHY5 overexpression lines and 3261 differentially expressed genes were screened in SmHY5 RNAi lines.The results of KEGG(Kyoto Encyclopedia of Genes and Genomes)pathway analysis of differentially expressed genes showed that the number of genes related to metabolism was the largest among the differentially expressed genes.The differentially expressed genes related to metabolism reached 52.23%and 50.24%of the total annotated genes in SmHY5 overexpression and SmHY5 RNAi lines,respectively.The differential expression of key enzyme genes in biosynthetic pathway of phenolic acids,tashinones and related genes in the IAA signaling pathway were analyzed.The results showed that SmHY5 increased the expression of several key enzyme genes in the biosynthetic pathways of the two type of components.SmHY5 affects the expression of key genes in many aspects of IAA signaling,such as IAA activity regulation,IAA polar transport and IAA signal transduction.It provides a rationale to understand the regulatory mechanism of SmHY5 on lateral root development.6.The target genes of SmHY5 in the biosynthetic pathway of phenolic acids were screened by yeast one-hybrid technique.The results show that SmHY5 can directly bind to the promoters of SmPAL1 and SmHPPR1,and regulate these two genes to affect the biosynthesis of phenolic acids.