基于H2DCFDA荧光探针的植物活性氧检测方法

doi:10.11983/CBB22043

摘要/Abstract

摘要： 活性氧(reactive oxygen species, ROS)是植物体内的一把“双刃剑”。ROS作为信号分子在植物生命活动中发挥关键作用, 但ROS过量积累会对生物大分子造成氧化损伤。准确测定ROS含量对于评估植物细胞内的氧化还原状态至关重要。由于植物体内ROS各组分半衰期短且反应活性强, 定性定量检测较为困难。因此, 选择合适的检测方法以提高检测的时空准确性非常重要。目前, 荧光分析法因其具有灵敏度高、选择性好、检出限低和直观性强等优点, 受到研究人员的广泛关注。该文详细描述基于流式细胞仪和激光共聚焦显微镜, 利用2′,7′-二氯二氢荧光素二乙酸酯(H2DCFDA)荧光探针检测水稻(Oryza sativa)体内ROS水平和时空分布的操作流程及注意事项。该技术也可用于直接检测拟南芥(Arabidopsis thaliana)、玉米(Zea mays)和大豆(Glycine max)等模式植物组织中ROS的水平和分布。

关键词: 活性氧, 荧光探针, H2DCFDA, 植物

Abstract: Reactive oxygen species (ROS) are a ‘double-edged sword’ in plants. On the one hand, ROS, as a signal molecule, plays pivotal roles in many aspects of life activities; on the other hand, excessive accumulation of ROS can cause oxidative damage to biological macromolecules. Accurate detection of ROS is essential to assess its intracellular redox status. Due to the characteristics of short half-life and strong reactivity of ROS components, their qualitative and quantitative analysis are difficult. It is critical to select the appropriate detection method and improve the spatiotemporal accuracy of detection for research in plant sciences and in other fields. At present, fluorescent probe analysis has attracted the attention of researchers because of its advantages of high sensitivity, good selectivity, low detection limit and strong intuition. This article introduces the detailed operation protocol and attentions for ROS detection using 2′,7′-dichlorodi-hydrofluorescein diacetate (H2DCFDA) fluorescent probe based on flow cytometry and confocal microscope. These methods can be used to detect ROS levels and distribution in model plant tissues, including Oryza sativa, Arabidopsis thaliana, Zea mays and Glycine max.

Key words: reactive oxygen species, fluorescent probe, 2',7'-dichlorodi-hydrofluorescein diacetate, plant

胡海涛, 钱婷婷, 杨玲. 基于H2DCFDA荧光探针的植物活性氧检测方法. 植物学报, 2022, 57(3): 320-326.

Haitao Hu, Tingting Qian, Ling Yang. Detection of Reactive Oxygen Species Using H2DCFDA Probe in Plant. Chinese Bulletin of Botany, 2022, 57(3): 320-326.

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链接本文: https://www.chinbullbotany.com/CN/10.11983/CBB22043

https://www.chinbullbotany.com/CN/Y2022/V57/I3/320

图/表 2

图1 基于2′,7′-二氯荧光素(DCF)的流式细胞仪检测水稻叶片原生质体活性氧含量 (A) 正常生长水稻叶片中的活性氧(ROS)荧光强度; (B) PEG-8000处理组水稻叶片中的ROS荧光强度; (C) 对照和处理组水稻叶片中的ROS相对荧光强度, 其值用于评估ROS含量。n=3; **表示经Student’s t检验在P<0.01水平差异显著。SSC: 侧向散射光

Figure 1 Reactive oxygen species (ROS) evaluation in rice protoplasts using 2′,7′-dichlorofluorescein (DCF)-based flow cytometry (A) ROS fluorescence intensity in rice leaves under normal growth conditions; (B) ROS fluorescence intensity in rice leaves under PEG-8000 treatment conditions; (C) The relative fluorescence intensity in treatment and control groups was determined to assess the ROS content. n=3; ** indicates significant difference at P<0.01 level by Student’s t test. SSC: Side scatter

图2 正常生长(A)-(D)和PEG-8000处理(E)-(H)的水稻叶片共聚焦荧光成像图红色为叶绿素的自发荧光, 绿色为2′,7′-二氯二氢荧光素二乙酸酯(H2DCFDA)氧化产生的2′,7′-二氯荧光素(DCF)荧光。Bars=100 µm

Figure 2 Confocal imaging analysis of rice leaves under normal growth (A)-(D) and PEG-8000 treatment (E)-(H) Red is the spontaneous fluorescence of chlorophyll, and green is the fluorescence of 2′,7′-dichlorofluorescein (DCF) generated by 2′,7′-dichlorodi-hydrofluorescein diacetate (H2DCFDA) oxidation. Bars=100 µm

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