Cloning And Functional Analysis Of EIL Transcription Factor Gene EIN3 From Salvia Miltiorrhiza

Salvia miltiorrhiza is a perennial herb whose roots can be used as medicine.The main active components of S.miltiorrhiza are lipid-soluble tanshinone and water-soluble salvianolic acid.Tanshinone belongs to diterpene and has a good role in promoting antisepsis,anti-inflammation,anti-tumor and anti-oxidation.While salvianolic acid B is one of the most well-known antioxidant natural products and it has good curative effect on cardiovascular and cerebrovascular diseases.The contradiction between its broad application prospects and shortage of supply make it a hot research topic in metabolic engineering.A 1797 bp transcription factor SmEIN3 is obtained by performing a local blast in the transcriptome library of Salvia miltiorrhiza according to AtEIN3.A preliminary study of its function is conducted by using technical measures,such as gene cloning,phylogenetic analysis,temporal and spatial expression pattern analysis,genetic transformation and functional verification,regulation and interaction analysis.The current results are as follows:1.We find SmEIN3 with a total length of 1797bp in the Salvia miltiorrhiza library and clone it.2.Phylogenetic tree analysis shows that SmEIN3 and SiEIN3 have the closest genetic relationship,followed by SmEIL1,AtEIN3 and AtEIL1.3.Tissue expression profile shows that the expression of SmEIN3 gene is the highest in leaf,followed by petiole,while the lowest in lord root and fibrous root.After induced by ABA,GA,SA,MJ,H₂O,ethanol,YE and ET,induced expression profile shows the expression of SmEIN3 is responsive to MJ and SA.With MJ and SA treatment,the expression can both reach the highest level at 12h.4.Using transient tobacco expression system to observe sub-cellular localization of SmEIN3 protein.The result shows that SmEIN3 protein localizes in the nucleus.This is consistent with the website's prediction and its feature as a transcription factor.5.The Sm EIN3 over-expressed vector and the suppressively expressed vector are constructed and the C58C1 agrobacterium is transformed to infect the explants of Salvia miltiorrhiza,and stably heritable hairy roots are obtained.Genomic PCR identification of 11 over-expressed lines and 3 suppressively expressed strains.We detect key enzyme genes of tanshinone pathway by qRT-PCR analysis and find the expression level of SmDXSII,SmCYP76AH1,SmDXR,SmKSL,SmGGPPS,SmHMGS,SmAACT,SmHMGR,SmCPS genes in tanshinone pathway are up-regulated in transgenic over-expression strains and down-regulated in the suppressively expressed strains.The expression level of Sm4CL1,SmCYP98A14,SmHPPR,SmRAS genes in salvianolic acid pathway are up-regulated in transgenic over-expressed strains while down-regulated among SmPAL,SmC4H and SmTAT.The expression level of these genes in suppressively expressed strains are just the opposite to over-expressed strains.6.Yeast one hybrid experiment shows that SmEIN3 can bind to EBS box element in the promoter of SmHPPR and SmKSL genes,and Dual-luciferase assay shows that SmEIN3 can positively regulate SmDXS2,SmHMGR and can negatively regulate the promoter of the SmTAT gene.Yeast two hybrid assay shows that SmEIN3has interaction with SmJAZ1,SmJAZ2L1 and SmMYC2.7.HPLC detection of tanshinone and salvianolic acid content in SmEIN3transgenic hairy roots,the results show that compared with the control,over-expression of SmEIN3 can increase the content of tanshinone and salvianolic acid while the situation in the suppressively expressed strains is just the opposite,indicating that over-expression of SmEIN3 gene can both promote the accumulation of tanshinone and salvianolic acid.8.The SmEIN3 gene is transferred to the model plant Arabidopsis thaliana,in which we can observe the senescence and non-convulsive phenotype in the leaf of transgenic Arabidopsis.Over-expression of SmEIN3 can increase the content of tanshinones and salvianolic acid metabolites in Salvia miltiorrhiza.Dual-luciferase experiment shows that SmEIN3 can directly activate the promoters of key enzyme genes such as HMGR and DXSII,and the yeast two-hybrid result shows that SmEIN3 interacts with SmMYC2 and SmJAZs.The effect suggests that the ethylene signaling pathway may interact with the JA signaling pathway to regulate the synthesis of secondary metabolites in Salvia miltiorrhiza.