生物技术通报2025，Vol.41Issue(9)：44-53,10.DOI:10.13560/j.cnki.biotech.bull.1985.2025-0222
植物和细菌TurboID邻近蛋白标记方法的建立
Establishment of TurboID Proximity Labeling Technology in Plants and Bacteria
摘要
Abstract
[Objective]TurboID-mediated proximity labeling technology is a novel protein-protein interaction research tool based on engineered biotin ligase,offering advantages including rapid labeling kinetics,high spatio-temporal resolution,and minimal cellular toxicity.However,its application remains limited to a few model species during current preliminary implementation stages.In this study,we developed TurboID expression systems in multiple representative species-monocots,dicots and prokaryotes-to systematically evaluate their labeling efficiency and applicability across evolutionary distant organisms.[Method]Recombinant vectors expressing"epitope tag-TurboID"fusion proteins were constructed for monocots(Oryza sativa),dicots(Arabidopsis thaliana,Solanum lycopersicum,and Nicotiana benthamiana),and bacteria(Escherichia coli),followed by transformation into respective species.Total proteins were extracted after biotin incubation,and fusion protein expression was verified via epitope tag-specific Western blot.Biotinylation of endogenous proteins was detected using streptavidin-HRP-based immunoblotting.[Result]Using ubiquitin promoters in plants and a T7 promoter in bacteria,we successfully achieved high-level expression of a"tag protein+TurboID"fusion protein in A.thaliana,S.lycopersicum,N.benthamiana,rice(Oryza sativa)callus,and E.coli.Western blot analysis confirmed robust expression of the fusion protein across all five systems.Following biotin treatment,multiple endogenous proteins were efficiently biotinylated in each system,indicating that the constructed TurboID system functioned effectively across monocots,dicots,and prokaryotes,demonstrating broad applicability and strong potential for further expansion.[Conclusion]A TurboID-based proximity labeling system is successfully established in monocotyledonous and dicotyledonous plants,as well as in bacteria,providing an efficient and reliable platform for analyzing complex protein interaction networks.关键词
TurboID/邻近蛋白标记/生物素/生物素连接酶/蛋白互作/拟南芥/水稻Key words
TurboID/proximity labeling/biotin/biotin ligase/protein-protein interaction/Arabidopsis thaliana/Oryza sativa引用本文复制引用
王芳,邵会茹,吕林龙,赵点,胡振,吕建珍,姜亮..植物和细菌TurboID邻近蛋白标记方法的建立[J].生物技术通报,2025,41(9):44-53,10.基金项目
山西省博士毕业生来晋工作奖励(SXBYKY2022112),杂粮种质资源创新与分子育种国家实验室(筹)项目(20220401090001-35) （SXBYKY2022112）
