基于凝胶固定原位可视化荧光分析法的根际微生物响应根系分泌物机制研究

Root exudates are important materials secreted by plant roots in order to efficiently activate soil nutrient and interact with soil microbes. However, traditional study methods of root exudates contain complex steps, accompanied with big errors and other defects. It is a very frustrating problem in study of pedology and related disciplines about how to collect, separate, purify and detect accurately the organic acids secreted by plant roots in situ. In order to take the advantages of quantum dots fluorescent probes in detection process, such as high-sensitivity, high accuracy and selected detection, especially, the fluorescent detection via probes can be carried out directly in complex system without separation and purification, moreover the fluorescent detection can be visualized in situ, this project will bring the fluorescent probe analysis method into the pedology research and build visual fluorescent gel membrane detection systems for roots secretion. We plan to spread out plants roots on gel membrane which loads carbon dots fluorescent probes, then the certain roots exudates (select citric acid, vanillin aldehyde as examples) and it concentration will be detected by fluorescence analysis method. With the advantages of the photoluminescent properties of carbon dots, this analysis method can not only avoid tedious procedure of roots exudates collection, separation and purification, but decrease detection error. In addition, the analytical investigation of microbial community structure changes at the corresponding fluorescence detection region of the gel membrane will be carried out. Thus, the response mechanism of microbial community diversity alteration to the roots exudates will be revealed. Moreover, the effectiveness and applicability of this new research method will be tested by comparing the detection results obtained by the classical method.

根系分泌物是植物根系高效活化土壤养分，最终影响根际微生物类群结构的重要物质。然而其传统研究方法步骤繁琐、耗时费力、误差大。如何准确收集、分离纯化和检测根系特定部位分泌物，一直是土壤学等相关学科亟需解决的技术难题。本课题拟借助量子点荧光分析法具有灵敏度高、精确性好和简便快捷的特点，不需要分离纯化即可在复杂体系中快速检测和原位可视化监测目标产物的优势，将荧光分析法用于土壤学研究，建立根系分泌物可视化凝胶膜印迹检测系统，将根系铺展在负载碳量子点荧光探针的凝胶膜上，进行特定根系分泌物（以柠檬酸、香草醛为例）的荧光识别和浓度检测，利用碳量子点的光致发光性质，省去繁琐、误差大的分泌物分离纯化步骤，原位快速检测根系不同部位特定分泌物浓度；同时检测凝胶膜对应区域因受分泌物影响而产生的微生物群落结构变化，并通过和传统经典检测方法对比，考察将新检测法用于微生物对根际分泌物响应机制研究的检测效果和适用性。

根系分泌物是植物根系高效活化土壤养分的重要化合物，然而其传统研究方法步骤繁琐、耗时费力、误差大。如何准确收集、分离纯化和检测根系特定部位分泌物，一直是土壤学亟需解决的技术难题。本课题借助量子点荧光分析法具有灵敏度高、精确性好和简便快捷的优点，不需要分离纯化即可在复杂体系中快速检测和原位可视化监测目标产物的优势，将荧光分析法用于土壤学研究。项目执行过程中，我们完成了根系分泌物可视化凝胶膜印迹检测系统的建立，首先以根系分泌物绿原酸为代表，通过分子印迹手段，制备了可特异性显色和识别绿原酸的碳量子点荧光探针，并将其负载在透明的高分子凝胶膜上，该系统可以初步用于绿原酸的原位显色和识别。另外，我们还借助荧光分析方法，制备了可快速识别和检测土壤重金属（Cr）的高分子凝胶系统，它们可以特异性地结合铬离子并在紫外光照下发出特异性荧光，从而可以用于土壤铬污染程度的快速检测，本项快速检测土壤重金属污染的材料和方法已经获得了一项国家发明专利授权，此外我们也开展了原位缓释型有机酸对土壤微生物群落结构变化的检测研究，研究结果已发表在Chemical Engineering Journal,2021,414:128777上。在本项目的资助下，我们潜心开展了用于土壤领域的新材料、新检测手段以及重金属污染治理新方法等探索和研究，项目负责人以通讯作者共发表SCI一区论文８篇，包括在Chemical Engineering Journal（IF10.65）上发表论文3篇，项目负责人以第一发明人获得授权发明专利3项。