茶树荧光性绿斑病叶的自发荧光显微分析
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摘要
茶树荧光性绿斑病是一种全国范围内普遍发生的茶树成叶生理性病害,该病害典型症状表现为叶片下表皮局部凸起、呈绿色,且能发射黄绿色荧光。
为了以黄、绿色自发荧光为探针了解茶树荧光性绿斑病的发生规律,为黄色、绿色荧光物质的分离鉴定提供光谱学依据,以及了解荧光性绿斑病叶的生理状态,本课题以茶树正常叶片与荧光性绿斑病叶为材料,应用荧光显微技术、显微荧光光谱成像技术、激光扫描共聚焦显微技术、流式细胞术研究了茶树荧光性绿斑病叶中黄、绿色自发荧光的显微观察条件、发射光谱、发光部位和病叶内部游离钙离子的分布以及胞内pH。结果表明:
(1)在不同激发光照射下,茶树荧光性绿斑病叶能发射多种自发荧光,其中黄、绿色荧光显微观察的最佳条件为:蓝光激发,彩色和绿色单色光模式记录图像;在绿光、黄色范围有三个荧光峰,其波长分别为515、535和585nm。
(2)最初发出绿色、黄色荧光的病变部位是维管束鞘细胞,随后出现在部分海绵细胞、栅栏细胞和表皮细胞中,在这些病变细胞中,叶绿体解体,红色荧光强度减弱,黄色、绿色荧光强度大,同时因为多个病变细胞密聚在一起,从而产生较强的荧光亮点能被肉眼所观察到;黄色、绿色荧光在细胞中的发光位置定位于液泡。
(3)正常叶片的胞内pH平均值为5.35,病害叶片的胞内pH平均值为6.89;病害叶片胞内pH与病害程度成显著正相关,胞内pH的升高是黄绿色荧光逐渐增强的一个原因。
(4)正常叶片胞内游离钙离子浓度明显高于病叶胞内游离钙离子浓度,胞间存在高浓度钙离子分布区域;病叶中开始病变细胞钙离子分布极化严重,完全病变细胞内已不存在游离钙离子;细胞低温装载探针的效果好于室温装载。
The Fluorescent Green Spot Disease was a physiological disease of tea mature leaves occurred all over the state in China. The representative symptom of this disease was green spots with fluorescence and irregular in shape.
In order to investigate the rule of the disease occurrence and the spectra of yellow-greenish fluorescent compounds, the fluorescence microscopy, microscopic fluorescence spectral imaging, laser scanning confocal microscopy and flow cytometry were used in this research. This experiment covered the condition of microscopic observation, emission spectral characters of yellow-greenish spontaneous fluorescence, and its localization in the diseased tea leaves, the distribution of the free Ca2+ and intracellular pH. The results showed that:
(1) The diseased leaves could emit several kinds of spontaneous fluorescence by irradiating different lights, and the optimal microscopic condition for observing the yellow-greenish fluorescence was that the diseased leaves were excited by blue light and images recorded in multicolor and green color mode, in which there were three fluorescence peaks at 515 nm, 535 nm and 585 nm within the light range of yellow and green.
(2) The yellow-greenish fluorescence was firstly emitted in the sheath cell of vascular bundle and then in the barrier cells, sponge cells and the epidermis cells. In these diseased cells, The chloroplasts disaggregated, the intension of their red fluorescence weakened and the yellow-greenish fluorescence was bright. And many diseased cells congregated closely,so the bright fluorescence spots can be observed. The fluorescence located in the vacuole of the diseased cells.
(3) The average intracellular pH of the normal leaves was 5.35 and the diseased leaves was 6.89 and the intracellular pH of the diseased leaves increased as the disease aggravated. The enhancement of intracellular pH was one cause of intensification of yellow-greenish fluorescence.
(4) The concentration of intracellular free Ca2+ of the normal leaves was higher than the diseased leaves. There was area which has high concentration Ca2+. In the diseased leaves, the distribution of free Ca2+ polarized seriously in primary diseased cells and there was no free Ca2+ in the entirely diseased cells. It was better loading the probe under low temperature than at room temperature.
为了以黄、绿色自发荧光为探针了解茶树荧光性绿斑病的发生规律,为黄色、绿色荧光物质的分离鉴定提供光谱学依据,以及了解荧光性绿斑病叶的生理状态,本课题以茶树正常叶片与荧光性绿斑病叶为材料,应用荧光显微技术、显微荧光光谱成像技术、激光扫描共聚焦显微技术、流式细胞术研究了茶树荧光性绿斑病叶中黄、绿色自发荧光的显微观察条件、发射光谱、发光部位和病叶内部游离钙离子的分布以及胞内pH。结果表明:
(1)在不同激发光照射下,茶树荧光性绿斑病叶能发射多种自发荧光,其中黄、绿色荧光显微观察的最佳条件为:蓝光激发,彩色和绿色单色光模式记录图像;在绿光、黄色范围有三个荧光峰,其波长分别为515、535和585nm。
(2)最初发出绿色、黄色荧光的病变部位是维管束鞘细胞,随后出现在部分海绵细胞、栅栏细胞和表皮细胞中,在这些病变细胞中,叶绿体解体,红色荧光强度减弱,黄色、绿色荧光强度大,同时因为多个病变细胞密聚在一起,从而产生较强的荧光亮点能被肉眼所观察到;黄色、绿色荧光在细胞中的发光位置定位于液泡。
(3)正常叶片的胞内pH平均值为5.35,病害叶片的胞内pH平均值为6.89;病害叶片胞内pH与病害程度成显著正相关,胞内pH的升高是黄绿色荧光逐渐增强的一个原因。
(4)正常叶片胞内游离钙离子浓度明显高于病叶胞内游离钙离子浓度,胞间存在高浓度钙离子分布区域;病叶中开始病变细胞钙离子分布极化严重,完全病变细胞内已不存在游离钙离子;细胞低温装载探针的效果好于室温装载。
The Fluorescent Green Spot Disease was a physiological disease of tea mature leaves occurred all over the state in China. The representative symptom of this disease was green spots with fluorescence and irregular in shape.
In order to investigate the rule of the disease occurrence and the spectra of yellow-greenish fluorescent compounds, the fluorescence microscopy, microscopic fluorescence spectral imaging, laser scanning confocal microscopy and flow cytometry were used in this research. This experiment covered the condition of microscopic observation, emission spectral characters of yellow-greenish spontaneous fluorescence, and its localization in the diseased tea leaves, the distribution of the free Ca2+ and intracellular pH. The results showed that:
(1) The diseased leaves could emit several kinds of spontaneous fluorescence by irradiating different lights, and the optimal microscopic condition for observing the yellow-greenish fluorescence was that the diseased leaves were excited by blue light and images recorded in multicolor and green color mode, in which there were three fluorescence peaks at 515 nm, 535 nm and 585 nm within the light range of yellow and green.
(2) The yellow-greenish fluorescence was firstly emitted in the sheath cell of vascular bundle and then in the barrier cells, sponge cells and the epidermis cells. In these diseased cells, The chloroplasts disaggregated, the intension of their red fluorescence weakened and the yellow-greenish fluorescence was bright. And many diseased cells congregated closely,so the bright fluorescence spots can be observed. The fluorescence located in the vacuole of the diseased cells.
(3) The average intracellular pH of the normal leaves was 5.35 and the diseased leaves was 6.89 and the intracellular pH of the diseased leaves increased as the disease aggravated. The enhancement of intracellular pH was one cause of intensification of yellow-greenish fluorescence.
(4) The concentration of intracellular free Ca2+ of the normal leaves was higher than the diseased leaves. There was area which has high concentration Ca2+. In the diseased leaves, the distribution of free Ca2+ polarized seriously in primary diseased cells and there was no free Ca2+ in the entirely diseased cells. It was better loading the probe under low temperature than at room temperature.
引文
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