基于固相微萃取技术的黄芪根际土壤环境中化感物质原位分析应用研究

Due to the low content and complicated basal influence, the in situ analysis of allelochemicals in the soil environment has become a technical bottleneck for the development of allelopathy. In our previous research, ten secondary metabolites were isolated and identified from the rhizosphere soil of cultivated Astragalus hoantchy, 6 of them observably showed significant allelopathic and autotoxic activity. Based on these results, a solid-phase microextraction system with well selective adsorption property and helpful to achieve efficient extraction in situ will be prepared with graphene oxide modified by ionic liquids as coating material and inner wall of porous midheaven stainless steel tube as carrier support according to the three types of the allelochemicals of A. hoantchy. With coupling of the system to LC-MS, the allelochemicals in the rhizosphere soil of A. hoantchy will be analyzed in situ to approach their structure-activity relationships, release sources and dynamic pattern of the allelochemicals of A. hoantchy. By the relevance analysis of the release of allelochemicals and environmental and pathogenic factors, the allelopathy response and chemical defence mechanism of A. hoantchy will be made clear. The implementation of this project could develop the technology system of allelopathy, expand the application field of solid-phase microextraction and advance the technical development of interdisciplines in the area of life science. The achievements could provide the academic and technical support for human being to understand more about the life activities of plants and relative applied development.

由于超微含量以及复杂的基底效应，土壤环境中植物化感物质的原位分析已成为阻碍该领域发展的技术瓶颈。项目前期从栽培黄芪根际土壤中分离鉴定出10种次生代谢物，明确了其中6种化合物具有明显的化感和自毒化感作用。在此基础上，针对黄芪三类化感物质的结构类型，以具有不同功能基团离子液体修饰的氧化石墨烯为涂层，以多孔中空不锈钢管内壁为载体，设计制备具有良好选择性吸附且能实现现场原位萃取的植物化感物质固相微萃取装置，通过与液-质联用，开展黄芪根际土壤环境中化感物质的原位分析，明确黄芪化感物质的构效关系和释放途径与动态变化规律；通过黄芪化感物质释放与环境及致病因子之间相关性分析，揭示黄芪的化感作用响应机制与化学防御机制。项目的实施，能够完善植物化感作用研究技术体系，拓展固相微萃取技术的应用领域，促进生命领域相关交叉学科的技术发展，为人类认识自然界植物生命活动规律和相关的应用研发提供理论和技术支撑。

采用Hummers法，制备了氧化石墨烯；结合乌拉特黄芪化感物质的结构特点，选择5种具有不同结构类型的离子液体键合氧化石墨烯为涂层，以多孔中空不锈钢管内壁为载体，利用自组装技术制备了固相微萃取装置；界面结构表征及萃取性能考察结果表明，1-胺乙基-3-甲基咪唑双(三氟甲烷磺酰)亚胺盐键合氧化石墨烯制备的固相微萃取装置对黄芪化感物质具有良好选择性与高效富集性能；通过萃取条件优化，构建了固相微萃取联用液相色谱高效富集检测黄芪化感物质土壤原位分析技术体系。通过对不同生长时期根际土壤中化感物质检测，明晰了乌拉特黄芪根际土壤中化感物质的动态变化规律。结合化感活性评价，探讨了环木菠萝烷型三萜和其它化感物质的构效关系及其释放途径；通过土壤环境中化感物质及其降解产物定性定量分析，结合结构修饰及化感活性评价，印证了对羟基苯甲酸为乌拉特黄芪自毒化感物质对羟基苯甲醛的氧化产物，通过土壤降解作用减弱化感物质的自毒作用。通过室内单因子实验，开展了环境因子改变与乌拉特黄芪化感物质释放规律的相关性分析；结合自然生态条件下化感物质释放与病虫害发生相关性研究，结果显示，乌拉特黄芪根际土壤中对羟基苯甲醛的含量随微生物病害的发生呈正相关。乌拉特黄芪在面对病原物侵袭时，通过根系分泌大量对羟基苯甲醛进入根际土壤，以应对环境胁迫以及微生物病害的发生，从而揭示了黄芪化感作用响应及其化学防御机制。项目实施期间，共发表SCI论文10篇，中文核心期刊3篇，会议论文6篇，授权发明专利5件，申请发明专利3件，培养博士研究生4人，硕士研究生1人。