基于多肽组学的植物多肽激素组分离分析新技术与新方法研究

In order to develop new analysis platforms for the research of plant hormonomics, the project is proposed to develop novel techniques and methods to separate and detect polypeptide hormonome in plant samples, and the main research are focused on as follows: (1) to synthesize universal metal-organic frameworks, and then to develop novel methods by coupling low temperature microwave-assisted extraction-metal-organic framework-based solid phase extraction with LC-QTOF/MS for separation and detection of polypeptide hormonome; (2) to obtain polypeptide aptamers for recognition of hydroxyproline-rich polypeptide, phytosulfokine and rapid alkalinization factor based on the systematic evolution of ligand by exponential enrichment (SELEX) technique, to prepare monolithic immunoaffinity column by the corresponding polypeptide aptamer, and then to develop novel methods by coupling low temperature microwave-assisted extraction-monolithic immunoaffinity column with LC-QTOF/MS for separation and detection of polypeptide hormonome; (3) to synthesize aptamer-functionalized magnetic particles, and then to develop novel methods by coupling low temperature microwave-assisted extraction-aptamer-functionalized magnetic particles with LC-QTOF/MS for separation and detection of polypeptide hormonome; (4) to develop novel methods by coupling aptamer-functionalized quantum dots with microfluidic chip-fluorescence microscope for separation and detection of polypeptide hormonome in plant cells. The object of the project is to provide novel analytical platforms for studying molecular mechanics of plant hormones and finding and determining new plant polypeptide hormones by the proposed analytical techniques and methods in this research.

本项目针对当前植物激素组学分析技术平台构建所面对的难题，拟建立植物多肽激素组分离分析新技术新方法。主要研究内容：（1）合成通用性金属有机杂化材料，构建低温微波辅助萃取-金属有机杂化材料固相萃取-LC-QTOF/MS联用分离分析植物多肽激素组新方法；（2）以富羟脯氨酸多肽、植物硫素和快速碱化因子为代表，筛选多肽激素适配体，制备基于适配体的多肽激素组免疫亲和整体柱，并建立低温微波辅助萃取-免疫亲和整体柱-LC-QTOF/MS联用分离分析植物多肽激素组新方法；（3）合成适配体功能化磁性微球，建立低温微波辅助萃取-免疫亲和磁性微球-LC-QTOF/MS联用分离分析植物多肽激素组新方法；（4）建立量子点标记多肽激素适配体荧光探针-微流控芯片-荧光显微镜联用原位、动态检测细胞中植物多肽激素组新技术。总体目标是以新技术与新方法为研究植物多肽激素组的分子作用机理、发现和确认新的多肽激素提供新的检测平台。

构建植物多肽激素分离分析新方法与新技术对研究植物激素的分子作用机理、发现和确认新的植物多肽激素具有重要意义。项目按照原研究计划进行，具体研究结果为：（1）本项目合成了MIL-100 (Fe)、MIL-53 (Al)和MIL-101（Cr）等金属有机杂化材料，构建了低温微波辅助萃取-金属有机杂化材料固相萃取-LC-MS/MS联用分离分析植物多肽激素组新方法；（2）合成了一系列高内相乳液整体柱，并采用系统素适配体进行了功能化，建立了低温微波辅助萃取-适配体功能免疫亲和高内相乳液聚合物整体柱- LC-MS/MS联用分离分析植物多肽激素组新方法；（3）合成了磁性金属有机-无机杂化材料及氧化石墨烯功能化磁性微球，构建了低温微波辅助萃取-磁性微球固相萃取-LC-MS/MS联用分离分析植物多肽激素组新方法；（4）合成了系统素适配体功能化量子点荧光探针，并用于选择性识别植物多肽激素新方法。上述研究所构建的新方法为研究植物样品中多肽激素组提供了新的分离分析检测新平台。