Isolation And Founctional Exploration Of Two JAZ Transcription Inhibitors In Salvia Miltiorrhiza

Salvia miltiorrhiza Bunge is a traditional Chinese medicine,there are two key ingredients in S.miltiorrhiza,including the hydrosoluble phenolic acids functioning to relieve myocardial ischemia and liposoluble tanshinone(tanshinone I,tanshinone IIA,tanshinone IIB,Cryptotanshinone),normally used in the treatment of purulent infectious diseases and cardio-cerebral vascular disorder,both of which do a great job in medical treatment.JAZ(Jasmonate ZIM-domain,JAZ)is a type of inhabiting factor in Jasmonic acid(JA)signaling pathway.In our study,we took 12 reported Arabidopsis thaliana JAZs’c DNA as probe and conducted blast analysis based on the Salvia miltiorrhiza transcriptom data of our labotatory,9 SmJAZs were identified and names were given according to their similarity to At JAZs.Acdording to their response to MJ induction data got previously.We found that among those 9 SmJAZs,SmJAZ3-Like and SmJAZ8display an earlier and more intensive reaction to MJ.Therefore,we chose the two SmJAZs to conduct the following research,by molecular cloning strategry we finally isolated and cloned these two JAZ genes in Salvia miltiorrhiza Bunge.With bioinformatics analysis about the structure of SmJAZ3-like and SmJAZ8,we found that both of the JAZs contain the NT domain(N end),ZIM domain which includes TIFY motif,NLS domains,and Jas domains(C end).Tobacco instantaneous transformation showed that SmJAZ3-Like preferentially expressed in the nucleus with less powerful sign detected on cytomembrane.While SmJAZ8 totally localized in the nucleus,tissues expression profile analysis showed both SmJAZ3-Like and SmJAZ8expressed in every part detected,with highest expression level of SmJAZ3-Like found in phloem,followed by fibrous root,stem and taproot;SmJAZ8 expressed highest in xylogen,followed by phloem stem and fibrous root.Induction expression profiles experiment showed that both SmJAZ3-Like and SmJAZ8 can be greatly influenced by those induction factors including MJ,YE,ABA,GA3,SA,though a lot of differences existed.Additionally,theplantover-expressionvectorsof p CAMBIA2300~+-SmJAZ3-Like,p CAMBIA2300~+-SmJAZ8 were successfully constructed.The plasmids of the vectors were introduced into A.tumefaciens strain C58C1.Finally,9 SmJAZ3-Like overexpression lines were attained and 9 SmJAZ8overexpression lines were obtained.q RT-PCR was applied to study the expression level of the key enzymes of tanshinones and Salvanic acid synthesis pathway,the results showed that SmJAZ3-Like had an obvious inhibition effect on Sm KSL and Sm DXR,which were the key enzymes involved in tanshinones synthesis pathway,meanwhile SmJAZ8 was found down-regulated Sm GGPPS,Sm KSL respectively;As to Salvanic acid synthesis pathway,we detected that SmJAZ3-Like had a greater inhibiting effect on Sm4CL1 and Sm C4H,however,SmJAZ8 had an obvious decreasing regulation on Sm C4H and a slight inhibitive effect on Sm RAS and Sm HPPR at the same time.In order to understand the complete function of these two JAZs in secondary metabolites production,we used HPLC to determine the tanshinones and salvanic acid accumulation,as the result showed that,over expression of SmJAZ3-Like led to the decrease of final tanshinones outcome by a small extent,the average tanshinones content is 2.34 mg/g DW in SmJAZ3-Like(OE).While over expression of SmJAZ8 decreased tanshinones production by a less extent,and 2.92 mg/g DW was obtained,less than 3.14 mg/g accumulated in the control.Next,we conducted parrallel exploration with salvanic acid.According to our data,none of SmJAZ3-Like overexpression lines could significantly influence the final salvanic acid accumulation in Salvia miltiorrhiza,however,this is not the case with SmJAZ8,which could down-regulate the outcome of salvanic acid to 32.4 mg/g averagely by overexpression,meaning that SmJAZ8 is a repressing regulator in salvanic acid synthesis pathway.In conclusion,we successfully cloned two S.miltiorrhiza JAZs genes,SmJAZ3-Like/SmJAZ8,and investigated their role in tanshinones and salvanic acid synthesis pathway,detected expression level of some key enzymes participating in the accumulation of secondary metabolite in Salvia miltiorrhiza.Our experiment have provided a deep understanding of these two JAZs’biological functions,which would be helpful to uncover the metabolic mechanism of tanshinones and Salvanic acid synthesis and provide a theoretical bedstone to improve S.miltiorrhiza quality.