微分干涉差显微成像方法高通量分析单个苏金云芽孢杆菌HD-1芽孢萌发 -- 分析化学,):
引用本文：
陈越, 彭立新, 王晓春, 黎永青, 刘军贤, 王桂文. 微分干涉差显微成像方法高通量分析单个苏金云芽孢杆菌HD-1芽孢萌发[J]. 分析化学, ):
CHEN Yue, PENG Li-Xin, WANG Xiao-Chun, LI Yong-Qing, LIU Jun-Xian, WANG Gui-Wen. High-throughput Investigation of Germination of Individual Bacillus Thuringiensis HD-1 Spores by Differential Interference Contrast Microscopy Imaging[J]. Chinese Journal of Analytical Chemistry, ):
微分干涉差显微成像方法高通量分析单个苏金云芽孢杆菌HD-1芽孢萌发
陈越1,2, 彭立新2, 王晓春2, 黎永青3, 刘军贤1, 王桂文2
1 广西师范大学物理科学与技术学院, 桂林 541004;
2 广西科学院生物物理实验室, 南宁 530007;
3 美国东卡罗来那大学物理系, 美国北卡罗来那州格林维尔NC 27858
苏云金芽孢杆菌(Bt)休眠的芽孢具有极强的抗逆境能力,在合适的条件下很快萌发。本研究采用倒置显微镜建立微分干涉差(DIC)显微成像方法,高通量实时监测大量单个HD-1芽孢的萌发过程,并运用拉曼光谱进行对比。结果显示,萌发过程DIC图像亮度变化动态与芽孢吡啶二羧酸的含量变化有对应性; 6 s的时间分辨率即可较好地呈现芽孢的萌发动态;以10 mmol/L丙氨酸为营养萌发剂,Bt芽孢在Tris-HCl(pH 8.3)和Hepes(pH 7.4)缓冲液中的萌发动态基本相同;Bt芽孢在非营养萌发剂月桂胺中萌发极其缓慢,而在外源2,6-吡啶二羧酸钙下萌发极其迅速。结果表明,应用DIC显微成像技术可实现高通量实时观测大量单个Bt芽孢萌发的动态过程,为了解Bt芽孢的萌发机制和异质性提供了极大便利。
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High-throughput Investigation of Germination of Individual Bacillus Thuringiensis HD-1 Spores by Differential Interference Contrast Microscopy Imaging
CHEN Yue1,2, PENG Li-Xin2, WANG Xiao-Chun2, LI Yong-Qing3, LIU Jun-Xian1, WANG Gui-Wen2
1 College of Physics Sciences & Technology, Guangxi Normal University, Guilin 541004, C
2 Laboratory of Biophysics, Guangxi Academy of Sciences, Nanning 530007, C
3 Department of Physics, East Carolina University, Greenville, NC 27858, USA
Bacillus thuringiensis(Bt), a unique bacterium which forms parasporal crystal protein and spore in parallel, is the most widely used environmentally in compatible biopesticide worldwide. Bt spore has a strong resistance to stress and adversity, and germinates rapidly once conditions are favorable for growth. In this work, differential interference contrast(DIC) microscopy was used to monitor the germination of individual Bt HD-1 spores, and the germination process was also investigated with Raman spectroscopy simultaneously. Experiment results showed that the decrease in normalized DIC intensity of Bt spore was consistent with the release of pyridine-2,6-pyridine dicarboxylic acid(dipicolinic acid[DPA]) determined by Raman spectroscopy. Temporal resolution of 6 seconds could well describe the dynamic process of spore germination. Bt spore showed similar germination kinetics triggered either by 10 mmol/L alanine at 37℃ in 25 mmol/L Tris-HCl buffer(pH 8.3) or in 25 mmol/L Hepes buffer(pH 7.4). Most spores did not germinate triggered by dodecylamine but germinated rapidly triggered by exogenous Ca-DPA. These results indicated that DIC microscopy imaging could be used to observe germination of individual Bt spores in real-time quantitatively, and help to the understanding of mechanisms of Bt spore germination and its heterogeneity.
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DOI: 10.11895/j.issn.0345
基金项目: 本文系国家自然科学基金项目(Nos.60035)资助
通讯作者: 刘军贤, 王桂文
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