Analysis Of TIFY Gene Family Of Salvia Miltiorrhiza And Its Interaction Protein Screening

Salvia miltiorrhiza Bunge,a perennial herb,belongs to the genus Salvia of the Labiatae.Its dried root and rhizome are used as well-known traditional Chinese medicine herb,also known as red root,red ginseng,blood ginseng and so on.As we all know,S.miltiorrhiza has anti-bacterial,anti-inflammatory,anti-cirrhosis and multiple pharmacological activities.The active ingredients of S.miltiorrhiza are divided into two main groups:lipid-soluble tanshinones(tanshinone IIA,cryptotanshinone,etc.)and water-soluble phenolic acids(caffeic acid,danshensu,rosmarinic acid,salvianolic acid B,etc.).A number of studies showed that when plants were subjected to biological or non-biological stress,they could synthesize plant hormones quickly.And then through the corresponding signal transduction system,plants could induce the synthesis of secondary metabolites to resist external stimuli.We found that jasmonic acid and its derivatives(such as methyl jasmonate)could efficiently induce the accumulation of tanshinone and salvianolic acid.However,it is unclear that how the jasmonic acid signal is identified and transduced to regulate the synthesis of secondary metabolites of S.miltiorrhiza.The core link and molecular basis during the jasmonic acid signal transduction pathway need to be further studied.TIFY family genes,as a plant-specific gene family,have been studied in several species.It is found that the family members played an important role in various stress response and hormone signal transduction systems.Especially,JAZ protein,as a TIFY subfamily member,was not only the core role acting as a repressor releasing the target transcriptional factors in jasmonic acid signal transduction pathway,but also the central hub of the role interacting with other pathway regulators in the complex hormone regulation network.However,it is unknown for the TIFY family genes structural characteristics and expression patterns in S.miltiorrhiza.Moreover,as the important members of TIFY family,JAZ proteins functions and their interaction factors are not clear in the signal transduction process.In this study,it is the first time for analyzing systematically the members of the SmTIFY family in the genome level.The interaction factors of JAZ proteins were screened initially by screening yeast two-hybrid library.The main results are as follows:1.We searched for the genomic database of S.miltiorrhiza by local BLAST to obtain all SmTIFY family genes firstly.And then we analyzed the basic characteristics of the selected SmTIFY genes and performed PCR.Finally,15 SmTIFY family members were identified,including 10 SmJAZ genes,3 SmZML genes,1 SmTIFY gene and 1 SmPPD gene.The SmTIFY family members contain at least one TIFY domain,in which the SmJAZ proteins have two conserved domains of TIFY and Jas.There are also EAR-like domains at the N-terminus of SmJAZ1,SmJAZ2,SmJAZ5 and SmJAZ6.SmZML proteins also have CCT domain and GATA zinc finger domain.The N-terminal of the SmPPD protein contains a PPD domain.While,SmTIFY8 only have one TIFY domain.2.The phylogenetic analysis of all TIFY family members from four species,including Arabidopsis thaliana,rice,grape and S.miltiorrhiza,found that all TIFY family members were clustered into four subfamilies by neighbor-joining phylogenetic tree.JAZ proteins from all species also were clustered into four subclasses for JAZ ?,JAZ ?,JAZ ?,and JAZ ?.3.The 2000 bp sequences of SmTIFY genomic DNA upstream of the initiation codon(ATG)were selected as the promoter sequences to analyze the cis-elements of SmTIFY genes,The results showed that the species and distribution of cis-elements from SmTIFY family genes promoter regions were diverse,which mainly were related to hormone response,stress response,light response and growth and development process.4.The expression pattern of SmTIFY family genes in roots,stems,leaves and flowers from S.miltiorrhiza was analyzed.The relative expression levels of SmJAZ1,SmJAZ3,SmJAZ5,SinJAZ7,SmJAZ8 and SmTIFY8 were higher in different tissues.Moreover,there was no significant difference in the expression of the same gene at different tissues.By treating the tissue culture seedlings of S.miltiorrhiza with 100 ?M MeJA for different time periods,the expression of majority SmJAZ family genes were induced in 2 h by MeJA and kept the same increasing trend,while the expression peak of SmJAZ3,SmJAZ4 and SmJAZ9 were delayed differently from other SmJAZ genes.In contrast,MeJA inhibited the expression of TIFY,PPD and ZML family genes.5.By taking SmJAZ3-Jas as the bait protein,the interaction proteins SmMYC2 and SmWD40-61 protein were found by screening yeast two-hybrid library.And then the WD40 protein was used as the bait protein for screening yeast two-hybrid library.The proteins of TCP,YABBY,PYL8 and PYL4 were screened,belonging to the regulatory proteins involved in the process of flower development and ABA signal pathway regulators.It was speculated that SmJAZ3 might regulate the growth and development of flowers and played an important role in JA-ABA signal control network.In addition,SmJAZ8-Jas was used as a bait protein to obtain SmCYP716A89,GRAS,VOZ1,PsbR and PSI-LHCII proteins.SmCYP716A89 might be involved in the regulation of Danshen diterpenoid synthesis pathway.GRAS protein played an important role in GA signaling pathway and participated in the regulation of plant growth and development.VOZ1,PsbR and PSI-LHCII proteins played an important role in regulating flowering time and photosynthesis,respectively.It is suggested that SmJAZ8 may play a role in regulating the diterpenoid synthesis pathway,plant photosynthesis and flowering time.This study laid a foundation for the identification of SmTIFY family members and the functional study of SmJAZ subfamily.And the study further explored the research ideas of regulation mechanism about the active components synthesis during the jasmonic acid signaling pathway of S.miltiorrhiza.Moreover,these results will also provide a reference for other medicinal plants metabolic mechanism research.