植物学报 ›› 2022, Vol. 57 ›› Issue (3): 320-326.DOI: 10.11983/CBB22043
收稿日期:
2022-03-10
接受日期:
2022-05-11
出版日期:
2022-05-01
发布日期:
2022-05-18
通讯作者:
杨玲
作者简介:
* E-mail: yangl@zjnu.cn基金资助:
Haitao Hu, Tingting Qian, Ling Yang()
Received:
2022-03-10
Accepted:
2022-05-11
Online:
2022-05-01
Published:
2022-05-18
Contact:
Ling Yang
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。
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.
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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