测定植物胞内游离钠离子的研究进展

Progress of the study on determination of free sodium in plant cells

关键词:  细胞内游离Na⁺  植物  荧光指示剂  低温酯导入  离子浓度计算

Key words:  intracellular free sodium  plants  fluorescent indicators  ester loading under low temperature  calculating the ion concentration

摘要:

植物耐盐机制的研究一直是植物抗性研究的焦点。近年来,随着生物学不断发展和新荧光标记技术的运用,胞内钠离子测定逐渐应用于植物盐胁迫研究中。该文论述了以下三方面问题:(1)分别介绍了SBFI、Sodium Green和CoroNa Green三种钠离子荧光指示剂:SBFI是一种双激发波长指示剂,其激发波长是340 nm/380 nm,发射波长是500 nm; Sodium Green和CoroNa Green是单波长指示剂,其激发波长分别是507 nm和492 nm,发射波长分别是532 nm和516 nm;(2)比较了酯导入、酸导入、电穿孔和显微注射等几种常见荧光指示剂载入胞内方法的优缺点,重点介绍了一种无损伤低温抑制酯酶法:先将荧光指示剂在缓冲液中低温(4 ℃)处理2 h,随后回到常温(20 ℃)在不含荧光指示剂的缓冲液中孵育2 h;(3)阐述了胞内离子浓度计算公式,包括单波长测定公式、双波长比率测定公式。

Abstract:

The mechanism of salt tolerance in plants had been the focus of plant resistance research in the past years. As the development of biology and application of new fluorescent labeling technologies,determination of intracellular free sodium had been gradually applied to the study of salt tolerance in plants. This review discussed three points as follows:(1)Introduction of three fluorescent indicators of intracellular free sodium: SBFI, Sodium Green and CoroNa Green. SBFI was a kind of fluorescent indicator for excitation ratio measurements, its emission ratio detected at 500 nm when excited at 340/380 nm. Sodium Green and CoroNa Green were fluorescent indicators that lacked a significant shift in emission or excitation wavelength upon binding to Na⁺. Sodium Green and CoroNa Green could be detected at 532 nm and 516 nm when excited at 507 nm and 492 nm respectively;(2)Compared the advantages and disadvantages of the protocols of loading the fluorescent indicators into cells,including AM esters of the fluorescent probes,acid loading,electroporation and microinjection. A non-invasive loading of acetoxymethyl ester under low temperature was introduced: loading the fluorescent indicator into cells by incubating the cells in solution at 4 ℃ for 2 h followed by 2 h incubation in the dye-free solution at 20 ℃;(3)The measurement of the internal sodium concentration in cells was illustrated. The equation for measurement of fluorescence intensity that lacked a significant shift in emission or excitation wavelength was:[Na⁺]=K_d(F-F_min)/(F_max-F). Fluorescence intensity(F)was targeted fluorescence intensity. F_min was appropriate mixtures of low Na⁺,and F_max was appropriate maximum of high Na⁺. The equation for measurement of fluorescence intensity ratio was:[[Na⁺]=K_dQ(R-R_min)/(R_max-R). The ratio of fluorescence intensity(R)was the ration F1/F2 of the fluorescence intensity. F_min was the ratio of appropriate mixtures of low Na⁺,and F_max was the ratio of appropriate maximum of high Na⁺.