水稻(Oryza sativa L.)OsDOF23是植物特异性转录因子DOF(DNA binding with one finger)蛋白家族的一员,但其具体的生物学功能和调节机制不明.本研究以OsDOF23为研究对象,预测了OsDOF23蛋白的理化性质和结构特性,分析了OsDOF23基因的组织表达图谱,及其在水稻群体中的单核苷酸多态性(single nucleotide polymorphisms,SNP)和插入缺失标记(insertion-deletion,InDel).此外,成功克隆并在大肠杆菌(Escherichia coli)中表达纯化了OsDOF23蛋白的C末端,并以其为抗原制作了OsDOF23的多克隆抗体,用于后续的免疫沉淀实验.经免疫沉淀和液相色谱串联质谱技术检测分析,从水稻总蛋白中得到与OsDOF23共沉淀的蛋白质组信息.经筛选得到37个可能与DOF23互作的蛋白,对这些蛋白进行功能富集分析发现,从生物过程来说,其主要富集于蛋白质代谢和运输功能方面;从分子功能来说,其主要富集于核酸结合活性和转运活性方面.这些结果表明OsDOF23蛋白可能通过与蛋白质代谢、核酸结合和转运等相关功能蛋白互作发挥其生物学功能.
Structural and Functional Analysis of OsDOF23 and Mass Spectrometry Analysis of Its Binding Proteins in Oryza sativa L.
OsDOF23 in Oryza sativa L.is a member of specific plant DOF(DNA binding with one finger)transcription factor family,but its biological function and regulatory mechanism remains elusive.Here,a series of studies on OsDOF23 were performed.The physico-chemical properties and structural characteristics of OsDOF23 protein were predicted,The tissue expression profile of OsDOF23 gene and its single nucleotide polymorphism(SNP)and insertion-deletion(InDel)were analyzed.Further,the C-terminal of OsDOF23 was cloned and the protein was expressed and purified in Escherichia coli,based on which polyclonal antibody of OsDOF23 was generated for the following OsDOF23 immunoprecipitation assay.37 OsDOF23 interacting protein candidates were identified from rice total protein by immunoprecipitation and liquid chromatography-tandem mass spectrometry.The function enrichment analysis showed that,in terms of biological process,they were mainly enriched in protein metabolism and cellular transport.In terms of molecular function,they were mainly enriched in nucleic acid binding activity and transporter activity.These results suggest that OsDOF23 protein may play a biolog-ical role through binding with proteins involved in the protein metabolism,nucleic acid binding and transport processes.
Oryza sativa L.DOF proteinProkaryotic expressionLiquid chromatography-tandem mass spectrometry
NETL
NSTL
万方数据
