Shanghai Collaborative Innovation Center of Agri-Seeds, Joint Center for Single Cell Biology, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China; Ministry of Agriculture and Rural Affairs Key Laboratory of Gene Editing Technologies (Hainan), Institute of Crop Sciences and National Nanfan Research Institute, Chinese Academy of Agricultural Sciences, Sanya, Hainan 572024, China; Hainan Seed Industry Laboratory, Sanya, Hainan 572024, China.
Shanghai Collaborative Innovation Center of Agri-Seeds, Joint Center for Single Cell Biology, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China; Shanghai Center for Plant Stress Biology, CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai 201602, China.
Plant Commun. 2024 Nov 11;5(11):101040. doi: 10.1016/j.xplc.2024.101040. Epub 2024 Jul 20.
Understanding the behavior of endogenous proteins is crucial for functional genomics, yet their dynamic characterization in plants presents substantial challenges. Whereas mammalian studies have leveraged in locus tagging with the luminescent HiBiT peptide and genome editing for rapid quantification of native proteins, this approach remains unexplored in plants. Here, we introduce the in locus HiBiT tagging of rice proteins and demonstrate its feasibility in plants. We found that although traditional HiBiT blotting works in rice, it failed to detect two of the three tagged proteins, a result attributable to low luminescence activity in plants. To overcome this limitation, we engaged in extensive optimization, culminating in a new luciferin substrate coupled with a refined reaction protocol that enhanced luminescence up to 6.9 fold. This innovation led to the development of TagBIT (tagging with HiBiT), a robust method for high-sensitivity protein characterization in plants. Our application of TagBIT to seven rice genes illustrates its versatility on endogenous proteins, enabling antibody-free protein blotting, real-time protein quantification via luminescence, in situ visualization using a cross-breeding strategy, and effective immunoprecipitation for analysis of protein interactions. The heritable nature of this system, confirmed across T to T generations, positions TagBIT as a powerful tool for protein study in plant biology.
理解内源性蛋白质的行为对于功能基因组学至关重要，但在植物中对其进行动态表征仍存在巨大挑战。虽然哺乳动物研究已经利用了带有发光 HiBiT 肽的基因座标记和基因组编辑来快速定量天然蛋白质，但这种方法在植物中尚未得到探索。在这里，我们引入了水稻蛋白质的基因座内 HiBiT 标记，并证明了其在植物中的可行性。我们发现，尽管传统的 HiBiT 印迹在水稻中有效，但它未能检测到三个标记蛋白中的两个，这是由于植物中的发光活性低所致。为了克服这一限制，我们进行了广泛的优化，最终采用了一种新的荧光素底物，并改进了反应方案，使发光强度提高了 6.9 倍。这一创新导致了 TagBIT（HiBiT 标记）的发展，这是一种在植物中进行高灵敏度蛋白质表征的强大方法。我们将 TagBIT 应用于七个水稻基因，说明了它在内源性蛋白质中的多功能性，能够进行无抗体的蛋白质印迹、通过发光进行实时蛋白质定量、使用杂交策略进行原位可视化以及进行有效的免疫沉淀以分析蛋白质相互作用。该系统在 T 到 T 世代的遗传特性使其成为植物生物学中蛋白质研究的有力工具。
