丹参SmERF081转录因子基因克隆与靶基因鉴定

Gene cloning and target gene identification of transcription factor SmERF081 from Salvia miltiorrhiza

目的:克隆丹参(Salvia miltiorrhiza Bge.)转录因子SmERF081,分析SmERF081的靶基因。方法:以丹参转录组数据Unigene(c48961-g1)序列为参考,利用PCR扩增获得SmERF081基因序列;通过生物信息学预测SmERF081编码蛋白的理化性质、蛋白质结构等分子特征;使用MEGA 7软件构建系统进化树;对SmERF081进行异源表达和蛋白纯化;通过酵母单杂、双荧光素酶报告系统和化学发光法凝胶迁移实验(EMSA)鉴定SmERF081与SmCPS5启动子是否结合。结果:克隆得到SmERF081基因,cDNA全长453 bp(Genbank登陆号:OQ466089),编码151个氨基酸,相对分子质量16.46 kDa,等电点9.75。该蛋白不含信号肽,无跨膜区,其高级结构主要由无规则卷曲构成。进化树分析表明SmERF081与拟南芥At1G28360.1、丹参SmERF8同源关系最近。原核表达结果显示,经IPTG诱导后SmERF081在大肠杆菌中成功异源表达,并通过纯化获得目的蛋白。酵母单杂、双荧光素酶报告基因检测和EMSA确认SmERF081可与丹参SmCPS5启动子结合。结论:鉴定到丹参中一个新转录因子SmERF081,生物信息学分析及分子互作初步证实其靶基因为丹参SmCPS5二萜环化酶。

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:OQ466089),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.