基于前期工作新发现和领域研究前沿，本申请项目旨在获得实验证据、阐明共生特异表达的紫云英转脂蛋白LTP基因AsE246在根瘤菌侵染和共生体发育中的互作机制。拟从以下两条途径展开研究，其一，基于免疫共沉淀偶联质谱技术和细菌双杂交技术，筛选鉴定与AsE246互作的华癸中慢生根瘤菌效应蛋白（effector protein）；进一步采用pull-down、双分子荧光互补(BiFC)、免疫荧光、基因插入失活等技术方法，确证二者体内外互作、共定位、以及效应蛋白基因的共生表型。其二，基于共生体（膜）分离、高效液相色谱偶联质谱技术，分析鉴定共生体膜中的特定脂质组分，并确证AsE246与特定脂质组分的内在联系。预期研究结果将为豆科植物-根瘤菌共生互作分子机理提供原创进展；也可为后续深入研究LTP/效应蛋白、脂筏、侵染线/共生体形成之间的功能机制奠定扎实基础。

This project is based on the new findings in our recent work and research advances in the field. It aims to obtain solid experimental evidences, and therefore to expound the symbiotic interacting mechanism of symbiotic-expressed lipid transfer protein (LTP) gene AsE246 of Astragalus sinicus in the rhizobial infection and symbiosome development process. The entire studies will be undertaken via the following two approaches. On the one hand, by using Co-Immunoprecipitation coupled with Mass Spectrometry and Bacterial Two-hybrid techniques, Mesorhizobium huakuii's effector protein as a specific binding partner for AsE246 will be screened and identified. The in vivo/vitro protein-protein interactions and co-localization for AsE246 and identified effector protein will be further investigated by using pull-down, bimolecular fluorescence complementation (BiFC), immunofluorescence (IF) techniques; the symbiotic phenotypes of the effector protein will be confirmed by the specific-locus insertion inactivation. On the other hand, by using symbiosome (membrane) isolation and high-performance liquid chromatography (HPLC)-Mass Spectrometry techniques, the lipid compositions of the M. huakuii pure culture, symbiosome and symbiosome membrane (SM) will be analyzed comparatively. The specific lipid component in the SM of plant origin and its relationship with AsE246 will be determined. The anticipated results will provide original research progress and contribution to the molecular mechanism for leguminous plant-rhizobia symbiotic interactions. Meanwhile, it can also establish a solid foundation to study in depth on the functional mechanism which involves in the LTP/effector proteins, lipid raft and infection thread/symbiosome formations in the future.