植物和细菌TurboID邻近蛋白标记方法的建立

doi:10.13560/j.cnki.biotech.bull.1985.2025-0222

摘要/Abstract

摘要：

目的 TurboID邻近蛋白标记技术是一种基于生物素连接酶的新型蛋白互作研究技术，具有标记速度快、时空分辨率高和毒性小等优点。目前该技术仍处于逐步推广阶段，并在少数物种中得到应用。通过构建多种代表性物种的TurboID表达系统，评估其在单子叶植物、双子叶植物及原核生物中的标记效能和应用范围。方法分别构建在单子叶植物［水稻（Oryza sativa）］、双子叶植物［拟南芥（Arabidopsis thaliana）、番茄（Solanum lycopersicum）、本氏烟草（Nicotiana benthamiana）］和细菌［大肠杆菌（Escherichia coli）］中表达“标签蛋白+TurboID”融合蛋白的载体，并将其转化至相应物种。通过生物素溶液处理转基因材料后提取总蛋白，利用标签蛋白免疫印迹检测融合蛋白的表达情况，并通过生物素免疫印迹检测内源蛋白的标记情况。结果在植物中采用泛素启动子、细菌中采用T7启动子驱动表达，实现了“标签蛋白+TurboID”融合蛋白在拟南芥、番茄、烟草、水稻愈伤组织和大肠杆菌中的高效表达。免疫印迹显示融合蛋白表达良好，且经生物素处理后，各系统中均有多种内源蛋白被有效标记，表明所构建的TurboID系统在单子叶植物、双子叶植物及原核生物中均具有良好适用性和推广潜力。结论成功在单子叶植物、双子叶植物和细菌中建立了TurboID邻近蛋白标记方法，为复杂蛋白互作网络分析提供了高效、可靠的技术平台。

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

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.

Key words: TurboID, proximity labeling, biotin, biotin ligase, protein-protein interaction, Arabidopsis thaliana, Oryza sativa

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.

WANG Fang, SHAO Hui-ru, LYU Lin-long, ZHAO Dian, HU Zhen, LYU Jian-zhen, JIANG Liang. Establishment of TurboID Proximity Labeling Technology in Plants and Bacteria[J]. Biotechnology Bulletin, 2025, 41(9): 44-53.

图/表 8

图1 植物和细菌TurboID邻近蛋白标记载体A：大肠杆菌TurboID邻近蛋白标记载体pDEST17-T7pro：：mCherry-6×His-TurboID；B：双子叶植物（拟南芥、番茄和烟草）TurboID邻近蛋白标记载体pK7-AtUBIpro：：3×HA-TurboID-EGFP；C：单子叶植物（水稻）TurboID邻近蛋白标记载体pK7-ZmUBIpro：：3×HA-TurboID-EGFP

Fig. 1 Expression vectors for TurboID proximity labeling in plants and bacteriaA: Expression vector (pDEST17-T7pro::mCherry-6×His-TurboID) for TurboID proximity labeling in Escherichia coli. B: Expression vector (pK7-AtUBIpro::3×HA-TurboID-EGFP) for TurboID proximity labeling in dicotyledonous plants, including Arabidopsis thalian, Solanum lycopersicum, and Nicotiana benthamiana. C: Expression vector (pK7-ZmUBIpro::3×HA-TurboID-EGFP) for TurboID proximity labeling in monocotyledonous plant (Oryza sativa)

图2 TurboID邻近蛋白标记技术原理示意图

Fig. 2 Schematic illustration of the TurboID proximity labeling technology principle

图3 大肠杆菌TurboID邻近蛋白标记方法的创建A：融合蛋白mCherry-TurboID的表达元件示意图；B：添加和不添加IPTG诱导剂的菌液照片；C：蛋白免疫印迹检测2个转基因大肠杆菌菌种（#1； #2）中融合蛋白的表达和邻近蛋白的生物素标记。“-”和“+”分别表示用ddH2O和50 μmol/L生物素溶液处理，在考马斯亮蓝染色图中分别表示未诱导和诱导后

Fig. 3 Establishment of the TurboID proximity labeling method in E. coliA: Schematic diagram of the expression elements of the fusion protein mCherry-TurboID3. B: Photographs of bacterial cultures with and without IPTG induction. C: Western-blot detection of the expression of the fusion protein and biotinylation of proximity labeling in two transgenic E. coli strains (#1; #2). "-" and "+" indicate treatment with ddH2O and 50 μmol/L biotin solution respectively, in the Coomassie Brilliant Blue staining image, they indicate the uninduced and induced states respectively

图4 邻近蛋白标记中生物素浓度与处理时间的优化A：使用不同浓度的生物素溶液对诱导后菌液处理3 h，使用α-AAL-Biotin和α-His抗体进行蛋白免疫印迹检测；B：使用50 μmol/L生物素溶液对诱导后菌液处理不同时间，进行蛋白免疫印迹检测

Fig. 4 Optimization of biotin concentration and labeling time used in proximity labelingA: Treat the induced bacterial culture with biotin solutions of different concentrations for 3 h and then perform Western-blot detection using α-AAL-Biotin and α-His antibodies. B: Treat the induced bacterial culture with a 50 μmol/L biotin solution for different durations and then perform Western-blot detection

图5 拟南芥TurboID邻近蛋白标记方法的创建A：融合蛋白TurboID-EGFP的表达元件示意图；B：野生型拟南芥与转基因拟南芥30 d幼苗图；C：蛋白免疫印迹检测2个转基因拟南芥株系（#3； #5）中融合蛋白的表达和蛋白的生物素标记。“-”和“+”分别表示用ddH2O和100 μmol/L生物素溶液处理。下同

Fig. 5 Establishment of the TurboID proximity labeling method in ArabidopsisA: Schematic diagram of the expression elements of the fusion protein TurboID-EGFP. B: Phenotypic comparison of 30-day-old seedlings between wild-type and transgenic Arabidopsis thaliana. C: Western-blot detection of the expression of fusion proteins and biotinylation of proximity labeling in two transgenic Arabidopsis lines (#3; #5). "-" and "+" indicate treatment with ddH2O and 100 μmol/L biotin solution respectively. The same below

图6 番茄TurboID邻近蛋白标记方法的创建A：野生型番茄与转基因番茄30 d幼苗图；B：蛋白免疫印迹检测2个转基因番茄株系（#1； #2）叶片融合蛋白的表达和邻近蛋白的生物素标记

Fig. 6 Establishment of the TurboID proximity labeling method in S. lycopersicumA: Comparative analysis of 30-day-old wild-type and transgenic Solanum lycopersicum. B: Western-blot detection of the expression of fusion proteins and biotinylation of proximity labeling in the leaves of two transgenic tomato lines (#1; #2)

图7 基于烟草瞬时表达的TurboID邻近蛋白标记方法的创建A：烟草叶片注射图片，Ruby表达农杆菌注射烟草作为烟草表达情况的可视化对照，CK为未注射农杆菌的叶片；B：蛋白免疫印迹检测融合蛋白的表达和邻近蛋白的生物素标记；从左到右3个样品分别是注射农杆菌但不注射生物素的叶片、未注射任何农杆菌的叶片和注射农杆菌且注射生物素（20 μmol/L）的烟草叶片

Fig.7 Establishment of the TurboID proximity labeling method based on tobacco transient expressionA: Image of tobacco leaf injection. Ruby-expressing Agrobacterium-injected tobacco serves as a visual control for tobacco expression, and CK being the leaf not injected with any Agrobacterium. B: Western-blot detection of the expression of fusion proteins and biotinylation of proximity labeling. From left to right, the three samples are tobacco leaves injected with Agrobacterium but not biotin, leaves not injected with any Agrobacterium or biotin, and tobacco leaves injected with both Agrobacterium and 20 μmol/L biotin

图8 水稻愈伤TurboID邻近蛋白标记方法的创建A：融合蛋白TurboID-EGFP的表达元件示意图；B：水稻愈伤图；C：蛋白免疫印迹检测2个转基因水稻株系（#1； #2）中融合蛋白的表达和邻近蛋白的生物素标记

Fig. 8 Establishment of the TurboID proximity labeling method in rice callusesA: Schematic diagram of the expression elements of the fusion protein TurboID-EGFP. B: Image of rice calluses. C: Western-blot detection of the expression of the fusion protein and biotinylation of proximity labeling in two transgenic rice lines (#1; #2)

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