Gene cloning and target gene identification of transcription factor SmERF081 from Salvia miltiorrhiza
Objective:To clone the transcription factor SmERF081 from Salvia miltiorrhiza Bunge and analyze its target gene.Methods:Using the UniGene(c48961-g1)sequence of S.miltiorrhiza transcriptome data as reference,the SmERF081 gene sequence was obtained by PCR amplification.Bioinformatics was used to predict the physicochemical properties,protein structure,and other molecular characteristics of the protein encoded by SmERF081.MEGA 7 was used to build the phylogenetic tree.Heterologous expression and protein purification on SmERF081 were performed.Whether SmERF081 binds to the promoter of SmCPS5(copalyl diphosphate synthase 5,CPS5)was identified by yeast one-hybrid system,dual-luciferase reporter system and EMSA.Results:The SmERF081 gene was cloned,with a total length of cDNA 453 bp(GenBank accession number:0Q466089),encoding 151 amino acids,the relative molecular mass of 16.46 kDa,and the isoelectric point of 9.75.The protein contains no signal peptide and no transmembrane region,and its higher-order structure is mainly composed of irregular curls.Phylogenetic tree analysis showed that SmERF081 had the closest homology with Arabidopsis At1G28360.1 and SmERF8.The results of Prokaryotic expression showed that SmERF081 induced by IPTG was successfully heterologously expressed in Escherichia coli and the target protein was obtained by purification.Yeast one-hybrid system,dual-luciferase reporter gene assay and EMSA verified that SmERF081 could bind to the promoter of SmCPS5.Conclusion:A new transcription factor SmERF081 in S.miltiorrhiza was identified.Bioinformatics analysis and molecular interactions preliminarily confirmed its target gene was SmCPS5 diterpene cyclase.
Salvia miltiorrhizaSmERF081bioinformaticstarget gene analysis
万方数据
维普
