Halo-tag标记技术及其在植物细胞成像中的应用

doi:10.11983/CBB22227

植物学报 ›› 2023, Vol. 58 ›› Issue (3): 475-485.DOI: 10.11983/CBB22227

Halo-tag标记技术及其在植物细胞成像中的应用

钱虹萍¹^,²^,³^,⁴, 罗鹏云¹^,²^,³^,⁴, 刘帅⁵, 徐昌文¹^,²^,³^,⁴, 殷金环¹^,²^,³^,⁴, 崔亚宁¹^,²^,³^,⁴(), 林金星¹^,²^,³^,⁴()

¹林木育种与生态修复国家工程研究中心, 北京 100083
²林木、花卉遗传育种教育部重点实验室, 北京 100083
³树木花卉育种生物工程国家林业和草原局重点实验室, 北京 100083
⁴北京林业大学生物科学与技术学院, 北京 100083
⁵中国林业科学研究院, 林木遗传育种全国重点实验室, 北京 100091

收稿日期:2022-09-29 接受日期:2023-01-28 出版日期:2023-05-01 发布日期:2023-05-17
通讯作者: *E-mail: cuiyaning@bjfu.edu.cn; linjx@bjfu.edu.cn
基金资助:
国家自然科学基金(32030010);国家自然科学基金(32000483)

Halo-tag Labeling Technology and It’s Application in Plant Living Cell Imaging

Hongping Qian¹^,²^,³^,⁴, Pengyun Luo¹^,²^,³^,⁴, Shuai Liu⁵, Changwen Xu¹^,²^,³^,⁴, Jinhuan Yin¹^,²^,³^,⁴, Yaning Cui¹^,²^,³^,⁴(), Jinxing Lin¹^,²^,³^,⁴()

¹Forest Tree Breeding and Ecological Restoration, National Engineering Research Center, Beijing 100083, China
²Key Laboratory of Genetics and Breeding in Forest Trees and Ornamental Plants, Ministry of Education, Beijing 100083, China
³The Tree and Ornamental Plant Breeding and Biotechnology Laboratory of National Forestry and Grassland Administration, Beijing 100083, China
⁴College of Biological Sciences and Biotechnology, Beijing Forestry University, Beijing 100083, China
⁵State Key Laboratory of Tree Genetics and Breeding, Chinese Academy of Forestry, Beijing 100091, China

Received:2022-09-29 Accepted:2023-01-28 Online:2023-05-01 Published:2023-05-17
Contact: *E-mail: cuiyaning@bjfu.edu.cn; linjx@bjfu.edu.cn

摘要/Abstract

摘要： 细胞是生物体的基本结构和功能单位, 其内部复杂的组织结构、相互作用及动力学过程等决定着整个生物体的生命形态和生命过程。开展活体状态下细胞结构和功能研究, 对于探索和掌握生命的本质具有重要意义。随着科学技术的不断进步, 一系列新的活细胞标记技术被不断革新。近年来, Halo-tag标记技术逐渐发展成为一种广泛应用的新型分子标记技术, 在活细胞成像和追踪研究领域取得了极大进展, 并逐渐在植物细胞成像中得到应用。该文首先系统介绍了Halo-tag标记技术的定义、分类和发展过程, 然后详细介绍了Halo-tag标记技术发生脱卤有机反应的机制及其荧光配基的种类, 最后从观察分子的精细定位、追踪分子的实时运动和检测分子间的相互作用3方面着重阐述了该技术在植物活细胞成像中的最新进展, 以期为后续Halo-tag标记技术在植物中的潜在应用奠定理论基础并提供技术支持。

关键词: 新型标记技术, Halo-tag标签, Halo-tag配基, 活细胞成像

Abstract: The cell is the basic structural and functional unit of living organisms, its complex internal organizational structure, interaction and process dynamics determine the life form and life process of the entire organism. The study of cell structure and function in the living state is of great significance for exploring and grasping the essence of life. With the continuous advances in science and technology, a series of new live cell labeling technologies have been innovated. In recent years, the Halo-tag labeling technology has gradually been developed into a widely used new molecular labeling technology, playing an increasingly important role in the field of living cell imaging analysis and tracking research. It has been gradually applied in plant cell imaging. In this review, we focus on the definition, classification and development process of Halo-tag technology, and introduce the reaction mechanism of the dehydrohalogenation of Halo-tag labeling technology and the types of fluorescent ligands in detail. Finally, we introduce the latest progress of this technology in the application of living cell imaging in plants is emphatically expounded from three aspects: observing the fine localization of molecules, tracking the real-time motion of molecules and detecting the interactions between molecules. Taken together, this study provides theoretical foundation and technical support for the potential application of the subsequent Halo-tag labeling technology in plants.

Key words: new labeling technology, Halo-tag label, Halo-tag ligand, living cell imaging

钱虹萍, 罗鹏云, 刘帅, 徐昌文, 殷金环, 崔亚宁, 林金星. Halo-tag标记技术及其在植物细胞成像中的应用. 植物学报, 2023, 58(3): 475-485.

Hongping Qian, Pengyun Luo, Shuai Liu, Changwen Xu, Jinhuan Yin, Yaning Cui, Jinxing Lin. Halo-tag Labeling Technology and It’s Application in Plant Living Cell Imaging. Chinese Bulletin of Botany, 2023, 58(3): 475-485.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB22227

https://www.chinbullbotany.com/CN/Y2023/V58/I3/475

图/表 2

图1 Halo-tag标记技术 (A) 活体细胞Halo-tag标记流程图; (B) 野生型(WT)和突变型脱卤酶的化学结构式(绿框表示脱卤酶的第106位天冬氨酸(Asp), 是指发生脱卤并形成中间产物酯的位点; 红框表示野生型与突变型脱卤酶的区别, 其中野生型脱卤酶第272位是组氨酸(His), 而在突变型脱卤酶第272位突变成苯丙氨酸(Phe)); (C) Halo-tag蛋白与TMR配基共价结合的分子模型(改自England et al., 2015)。

Figure 1 Halo-tag technology (A) Schematic showing Halo-tag labeling in living cells; (B) Chemical structural formulae of wild-type (WT) and mutant dehalogenases (green box indicates that aspartic acid (Asp) at position 106 of dehalogenase, which is the site where dehalogenation occurs and the intermediate ester is formed; red box indicates the difference between the wild-type dehalogenase and the mutant dehalogenase, where the wild-type dehalogenase is histidine (His) at position 272, while the mutant dehalogenase mutates to phenylalanine (Phe) at position 272); (C) Molecular model of the Halo-tag protein with a covalently bound TMR ligand (modified from England et al., 2015).

表1 用于活细胞成像的Halo-tag配基种类(改自Benink and Urh, 2015)

Table 1 Types of Halo-tag ligands for living cell imaging (modified from Benink and Urh, 2015)

配基类别	膜渗透性	激发/发射波长的峰值(nm)	是否需要清洗	化学结构式
TMR	细胞渗透性	555/585	是
diAcFAM	细胞渗透性	494/526	是
Oregon Green	细胞渗透性	496/516	是
Coumarin	细胞渗透性	353/434	是
Alexa Fluor? 488	非细胞渗透性	494/517	是
Alexa Fluor? 660	非细胞渗透性	663/690	是	结构图无法获得
TMRDirect?	细胞渗透性	555/585	否
R110Direct?	细胞渗透性	502/527	否

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Halo-tag标记技术及其在植物细胞成像中的应用

Halo-tag Labeling Technology and It’s Application in Plant Living Cell Imaging

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