植物病毒侵染的植物亚细胞内谷胱甘肽和活性氧的实时活体生物传感成像

项目名称： 植物病毒侵染的植物亚细胞内谷胱甘肽和活性氧的实时活体生物传感成像

项目编号： No.21305131

项目类型： 青年科学基金项目

立项/批准年度： 2014

项目学科： 数理科学和化学

项目作者： 雷荣

作者单位： 中国检验检疫科学研究院

项目金额： 25万元

中文摘要： 谷胱甘肽和活性氧是植物在抵御病原物侵染时起着重要信号传导作用的小分子化合物。现有分析方法都难以对两者进行实时、动态、灵敏的分析。为了研究植物病毒侵染和植物抗性机理，本项目拟用对氧化还原敏感的绿色荧光蛋白(redox GFP,roGFP)构建靶定于线粒体、叶绿体和细胞质的生物传感器Grx-GFP 和Prx-GFP,分别用于实时动态监测谷胱甘肽和过氧化氢。把此生物传感器转入植物，得到表达roGFP 的转基因烟草和拟南芥。以黄瓜花叶病毒为模式植物病毒，一方面可用于实时动态监测植物病毒侵染植物后，其体内的谷胱甘肽和活性氧随症状发展的产生情况，另一方面可用于筛选植物病毒真正引起黄化症状的基因。结合该病毒侵染过程中叶绿素的生物合成和叶绿体蛋白质变化的转录组和代谢组学分析，提出黄瓜花叶病毒的致病机理。本项目所构建的质粒和转基因植物材料还可用于其它病原物致病机理的研究，具有广阔的应用前景。

中文关键词： 活性氧；氧化还原绿色荧光蛋白；荧光寿命成像；转录组学；代谢组学

英文摘要： Both glutathione (GSH) and reactive oxygen species (ROS) play pivotal roles in defensing the pathogen infection. The present analysis methods cannot provide in vivo, real-time and dynamic information about this two small molecules. In order to investigate the plant virus infection and plant defense mechanism, we plan to design the fusion of glutaredoxin (Grx) and peroxiredoxins (Prx)，respectively to roGFP2 to facilitate specific real-time equilibration between the sensor protein and the glutathione redox couple and H2O2.The biosensor will be targeted to chloroplast, mitochondrial and cytosol with the help of some transit peptide, which will make the exact sub-cellular detection of GSH and ROS. Transgenomic tobacco and Arabidopsis expressing roGFP will be obtained with this biosensor plasmid. Combined the transcriptome and metabolomic analysis of the chlorophyll biosynthesis in different symptoms development, the cucumber mosaic virus infection mechanism based on redox is expected to present. Moreover, the transgentic plants expressing the biosensors can be used for the study of other pathogens such as bacterial, phytoplasma, fungi and other plant virus. This temporal resoluted and sensitive biosensor will facilitate the observation of redox changes associated with plant virus, bacterial and fungi infection.

英文关键词： Reactive oxygen species；redox green fluorescence protein；luorescence lifetime imaging；transcriptomics；metabolomics