《植物生理学报》 2021, 57(4): 963-968

荧光探针检测娃娃菜组织钙离子的条件优化

夏伦港^1,3，赵雅楠¹，任成斌¹，张也¹，李嘉靖¹，李雯琳¹，胡琳莉^1,2,*，郁继华^1,2,*

¹甘肃农业大学园艺学院, 兰州730070；²甘肃农业大学甘肃省干旱生境作物学重点实验室, 兰州730070；³华中农业大学园艺林学学院, 武汉430070

通信作者：胡琳莉；E-mail: hull@gsau.edu.cn；郁继华；E-mail: yujihua@gsau.edu.cn

摘　要：

植物体内Ca²⁺含量不足是引起娃娃菜干烧心病的主要原因之一, 为了探究温度对娃娃菜幼苗Ca²⁺吸收及分布的影响, 本研究以娃娃菜幼苗叶片及根系组织为试验材料, 将Ca²⁺荧光探针Fluo-3/AM载入娃娃菜细胞中, 并结合激光扫描共聚焦显微镜技术, 先优化荧光探针检测娃娃菜叶片及根系胞质游离Ca²⁺的测定方法, 然后应用优化的测定方法研究不同温度对娃娃菜幼苗Ca²⁺吸收及分布的影响。结果表明, 采用20 μmol·L^-1的Ca2+荧光指示剂Fluo-3/AM装载, 依次进行低温4°C加载90 min、 25°C去酯化90 min, 可获得较为理想的娃娃菜胞质游离Ca²⁺染色结果。不同温度处理7 d后, 高温胁迫下(35°C/20°C, 昼/夜)娃娃菜叶片中Ca2+含量与适宜温度下(25°C/18°C, 昼/夜)无明显差异, 低温胁迫下(10°C/5°C, 昼/夜)叶片组织Ca2+含量明显高于适宜温度; 根尖组织中Ca²⁺含量在三个温度处理下无明显差异。该试验方法可用于检测娃娃菜胞质游离Ca²⁺的变化, 为研究娃娃菜胞内钙信号变化及选育抗干烧心品种提供技术基础。

关键词：娃娃菜; Ca2+吸收; 荧光探针; 温度胁迫

收稿：2020-12-02   修定：2021-03-23

资助：甘肃农业大学学生大学生创新创业计划(201912044)、中央引导地方科技发展专项资金(ZCYD-2020-5)、甘肃农业大学公招博士 科研启动基金(GSAU-RCZX201713)和甘肃农业大学盛彤笙科技创新基金(GSAU-STS-2018-32)

Optimization of conditions for the determination of calcium ion in mini Chinese cabbage tissues by fluorescent probe

XIA Lungang^1,3, ZHAO Yanan¹, REN Chengbin¹, ZHANG Ye¹, LI Jiajing¹, LI Wenlin¹, HU Linli^1,2,*, YU Jihua^1,2,*

¹College of Horticulture, Gansu Agricultural University, Lanzhou 730070, China; ²Gansu Provincial Key Laboratory of Aridland Crop Science, Gansu Agricultural University, Lanzhou 730070, China; ³College of Horticulture and Forestry Sciences, Huazhong Agricultural University, Wuhan 430070, China

Corresponding author: HU Linli; E-mail: hull@gsau.edu.cn; YU Jihua; E-mail: yujihua@gsau.edu.cn

Abstract:

Insufcient calcium ion (Ca²⁺) in plants is one of the main reasons for tipburn occurrence in mini Chinese cabbage. The leaf and root of mini Chinese cabbage seedlings were used as test materials to explore the effect of temperature on Ca²⁺ absorption and distribution. The Ca2+ fluorescent probe Fluo-3/MA was loaded into mini Chinese cabbage cells, combined with laser confocal scanning microscopy technology, the fluorescence probe method of detecting the free Ca²⁺ in leaf and root was frstly optimized. Then the optimized method was applied to study the influence of different temperatures on Ca²⁺ absorption and distribution in mini Chinese cabbage seedlings. The results showed that the 20 μmol·L^-1 calcium ion fluorescent indicator Fluo-3/AM was loaded, followed by loading at 4°C for 90 min and de-esterifcation at 25°C for 90 min, an ideal Ca2+ staining result of mini Chinese cabbage could be obtained. After treated with different temperatures for 7 days, the calcium ion content of leaves under high temperature stress (35°C/ 20°C, day/night) had no obvious difference with that under suitable temperature (25°C/18°C, day/night), and the calcium ion content of leaf under low temperature stress (10°C/5°C, day/night) was signifcantly higher than that under the suitable temperature. The calcium ion content in the root tip tissue had no obvious difference among the three temperature treatments. The optimized method can be used to detect the change of free calcium ions in the cytoplasm of mini Chinese cabbage tissues, and it will provide a technical basis for the study of changes in intracellular calcium signal and the breeding of tipburn resistant varieties of mini Chinese cabbage.

Key words: mini Chinese cabbage; Ca2+ absorption; fluorescent probe; temperature stress