通过免疫共沉淀和蛋白质谱鉴定与拟南芥DSP1互作的蛋白  

Identification of Arabidopsis thaliana DSP1 Interacting Proteins via Immunoprecipitation and Protein Mass Spectrometry

作　　者：沈敏 徐道博 彭美玲 卢嘉怡 李一博 苏元 彭雪艳 刘云峰 SHEN Min;XU Daobo;PENG Meiling;LU Jiayi;LI Yibo;SU Yuan;PENG Xueyan;LIU Yunfeng(College of Life Science and Technology,Guangxi University,Nanning,530004)

机构地区：[1]广西大学生命科学与技术学院,南宁530004

出　　处：《基因组学与应用生物学》2024年第3期387-399,共13页Genomics and Applied Biology

基　　金：国家自然科学基金项目(31970603);广西自然科学基金(2019GXNSFDA185001);广西大学大学生创新训练项目(202110593065)共同资助。

摘　　要：拟南芥(Arabidopsis thaliana)DSP1(defective in snRNA processing 1)蛋白是DSP复合物的主要成员,其参与snRNA转录和剪切成熟,并在植物生长发育的多个方面发挥重要作用。本研究利用在线平台分析了DSP1蛋白的理化性质,并预测了DSP1蛋白的二级结构和高级结构。在全长DSP1蛋白无法在细菌表达系统中诱导的情况下,通过无缝克隆技术成功构建包含DSP1蛋白N端(DSP1-N)的重组蛋白表达载体,并且成功纯化了GST-DSP1-N重组蛋白,进一步制备了DSP1的特异性多克隆抗体。通过免疫共沉淀和液相色谱串联技术,共鉴定出45个潜在的与DSP1互作的蛋白,后续的功能聚类分析结果暗示DSP1蛋白可能通过蛋白质代谢、核酸结合活性等方面发挥生物学功能。本研究可为进一步揭示DSP1蛋白在植物中的功能提供研究目标和方向。Arabidopsis thaliana DSP1(defective in snRNA processing 1)protein is one of the essential member in DSP complex.It is involved in snRNA transcription and maturation and plays vital roles in multiple plant developmental processes.This study has taken advantage of online tools to predict the physical and biochemical properties of DSP1 and its secondary and tertiary structures.Since the attempt of expressing full-length DSP1 protein in bacteria was not successful,instead we were able to express and purify GST-DSP1-N fragment in N-terminus of DSP1 and further developed the polyclonal antibody of DSP1 using purified GST-DSP-N protein.Then a total of 45 candidate proteins were identified as the potential DSP1 interacting proteins by immunoprecipitation assay and liquid chromatography tandem technology.The functional clustering analysis of these candidates suggested that they may function in protein metabolism or nucleic acid binding processes.The study has revealed the future objectives and directions for further investigation of DSP1 functions.

关 键 词：DSP1蛋白 重组蛋白 免疫共沉淀 液相色谱-串联质谱法 互作蛋白 

分 类 号：Q946[生物学—植物学]