脂质转移蛋白基因AsE246在根瘤菌侵染和共生体发育中的共生互作机制研究

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.

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。

基于前期工作发现和领域前沿，本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。围绕这个目标，从两条途径展开研究内容，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功能蛋白；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、以及互作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，分析鉴定共生体中的特定脂质组分，并确证AsE246与特定脂质组分的内在联系。.通过项目实施，本项目首次分离、鉴定并获得了与脂质转移蛋白AsE246相互作用的根瘤菌蛋白htpG及编码基因的突变表型，获得了二者体、内外相互作用及亚细胞共定位的直接证据。分离鉴定了共生体膜特定脂质组分及其超显微定位，阐明了AsE246表达与特定脂质组分双半乳糖基甘油二脂（digalactosyldiacylglycerol）DGDG的内在联系。发现和阐明了DnaJ-like蛋白AsDJL1共生条件下与转脂蛋白AsE246相互作用，参与协助DGDG等脂质转运，在结瘤和共生固氮中发挥重要作用。从DGDG合成基因DGD1的角度，阐明了DGDG在根瘤共生体发育和固氮中的功能机制。基于上述发现，进一步首次提出了一个工作模型：脂质转移蛋白AsE246通过与华癸根瘤菌分子伴侣蛋白htpG、紫云英功能协助蛋白AsDJL1相互作用，构成一个蛋白机器复合体，共同介导豆科宿主植物合成脂质组分（特别是DGDG）转移至共生体膜中，进而调控根瘤菌侵染和共生体发育。.本项目取得的成果首次揭示了豆科植物根瘤中特异表达的脂质转移蛋白LTP基因，在根瘤菌侵染与共生体发育过程中的菌植互作机制，为豆科植物-根瘤菌共生固氮体系调控的分子机理研究提供了原创性进展。也为持续深入研究根瘤共生固氮中的脂质重塑和作用机制奠定了扎实基础。

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