Rapid and dynamic detection of endogenous proteins through in locus tagging in rice  

作　　者：Yifu Tian Dating Zhong Rundong Shen Xinhang Tan Chen Zhu Kai Li Qi Yao Xinbo Li Xuening Zhang Xuesong Cao Pengcheng Wang Jian-Kang Zhu Yuming Lu 

机构地区：[1]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 [2]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 [3]Shanghai Center for Plant Stress Biology,CAS Center for Excellence in Molecular Plant Sciences,Chinese Academy of Sciences,Shanghai 201602,China [4]Institute of Advanced Biotechnology and School of Medicine,Southern University of Science and Technology,Shenzhen 518055,China [5]Hainan Seed Industry Laboratory,Sanya,Hainan 572024,China

出　　处：《Plant Communications》2024年第11期14-22,共9页植物通讯（英文）

基　　金：National Key R&D Program of China(no.2021YFD1201300 to Y.L.and 2021YFA1300404 to J.-K.Z.);National Natural Science Foundation of China(32070396 to Y.L.);China Postdoctoral Science Foundation(BX20220098 and 2022M720973 to Y.T.).

摘　　要：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 T1 to T3 generations, positions TagBIT as a powerful tool for protein study in plant biology.

关 键 词：BREEDING FIR ENDOGENOUS 

分 类 号：Q942[生物学—植物学]