茶云纹叶枯病侵染叶片细胞壁多糖的拉曼化学成像机理及特性研究

The cell wall of higher plant which is mainly composed of polysaccharides, is the primary structural barrier of cell against pathogen infection. All plant pathogens interact with cell wall palysaccharides, so it is meaningful for detection of plant pathogenesis to monitor dynamic variation of cell wall palysaccharides during the plant pathogenic process. This project will investigate the Raman chemical imaging mechanism and characteristic of cell wall polysaccharides of tea leaf infected by tea leaf blight. Firstly, the Raman characteristic bands will be assigned to polysaccharides constitutes according to the Raman scattering rationale and the Raman spectroscopy of standard polysaccharides, and with the aid of chemometrics the obvious and specific Raman bands will be selected as quantitative analysis peaks. Then the approach will be developed for qualitative discrimination and quantitative determination of polysaccharides constitutes in cell wall. Furthermore, the topochemical distribution of polysaccharides constitutes in cell wall could be visualized by integrating microscopic image of cell wall with the Raman fingerprint bands which are contributed by polysaccharides constitutes. As a result the Raman chemical imaging approach will be developed for analysis of cell wall polysaccharides, and it will be used for detection of variation of cell wall polysaccharides during the pathogenic process. This research will lay a solid foundation for determination of other polymers in cell wall in situ quickly, and benefit the understanding of the plant-pathogen interactions.

主要由多糖类物质组成的高等植物细胞壁，是细胞抵抗病原物侵染的首要屏障，所有植物病原物在入侵细胞时均与细胞壁多糖发生作用，因此监测此过程细胞壁多糖的动态变化对于探明植物的发病机制具有重要意义。本项目以被茶云纹叶枯病侵染的茶树叶片为试验对象，研究染病叶片细胞壁多糖的拉曼化学成像机理及特性。首先依据多糖标准品的拉曼光谱指认染病组织细胞壁多糖成分的拉曼特征谱带，并借助化学计量学方法筛选出波峰明显且专一性好的谱带，作为多糖成分的定量峰，建立多糖成分的定性鉴别和定量检测方法；再结合细胞壁的显微图像信息绘制出多糖成分在细胞壁上的空间分布图，实现细胞壁多糖成分的定位检测，从而建立细胞壁多糖的拉曼化学成像检测方法；然后借助该方法探索茶云纹叶枯病侵染过程中细胞壁多糖的变化规律。本项目的研究结果将为细胞壁中其它多聚体的原位快速检测奠定坚实基础，并对进一步探索植物-病原物的分子互作机理具有重要意义。

本项目聚焦真菌与叶片互作下细胞壁多糖的原位化学成像分析。首次利用共焦显微拉曼技术对染病前后的龙井43茶树叶片的外观信息、内部化学成分和细胞结构进行无损检测，主要的结果如下：.⑴ 针对拉曼光谱中普遍存在的宇宙射线和荧光干扰问题，提出了一种能够自动识别宇宙射线的方法，提高了后续化学成像的效果，该方法已经获得发明专利授权。.⑵ 研究了云纹叶枯病菌菌丝体的拉曼光谱响应机理和特性，发现菌丝体中最显著的拉曼特征峰来源于几丁质的1622cm-1，基于几丁质的特征波段绘制了菌丝体的原位生长动态图，该研究成果已经在SCI收录期刊上发表。.⑶ 研究了健康茶叶和染病细胞壁的拉曼光谱响应特性，发现细胞壁染病后的拉曼特征峰有明显区别，对应果胶，纤维素和木质素这三类物质的峰强变化最为显著，其中果胶含量下降的最多，纤维素其次，木质素减少的相对较少。该研究成果已经在SCI收录期刊上发表。.⑷ 研究了不同染病阶段维管束组织细胞的拉曼光谱响应特性，发现随着染病的深入，维管束部位的纤维素和木质素的含量持续下降，尤其是木质素下降的量非常显著，表明共聚焦显微拉曼光谱能够实现染病组织细胞壁结构和成分的原位同步检测。.