根瘤细胞-共生体信号网络的蛋白时空图谱绘制及其中关键蛋白的作用机制解析

豆科植物-根瘤菌共生固氮过程中，根瘤菌进入根瘤细胞形成的类细胞器结构-共生体是共生固氮的基本结构单元。共生体的分化和代谢受到根瘤细胞的严格调控，然而根瘤细胞-共生体信号网络的蛋白时空图谱及其作用机制还有待研究。蒺藜苜蓿DNF1基因是内质网信号肽酶切复合体的组分，dnf1突变体中根瘤细胞-共生体分泌蛋白的信号肽不能被切除，共生体不能正常分化和固氮。因此，dnf1是研究根瘤细胞-共生体信号传递网络的重要工具。为揭示根瘤细胞-共生体信号网络的蛋白图谱及其调控机理，本课题将开展：1）提取野生型和dnf1突变体根瘤细胞内质网蛋白和共生体蛋白，进行蛋白质组iTRAQ测序；2）通过生物信息学分析，绘制根瘤细胞-共生体信号传递网络蛋白时空图谱；3）对信号网络中的关键蛋白进行功能分析，揭示其调控共生体分化的作用机理。本课题将从蛋白质组水平研究根瘤细胞-共生体信号传递网络，并解析其调控共生体分化的分子机制。

Legume-rhizobia nitrogen fixing symbiosis can provide plants with environment-friendly nitrogen source. The basic functional structure of nitrogen fixing symbiosis is symbiosome (a subcellular organelle-like structure formed by rhizobia inside nodule cells). The dynamic changes of symbiosome are regulated by the nodule cell-symbiosome signal transduction network, however the components, structure and functional mechanisms of this network remain to be unrevealed. Medicago truncatula DNF1 is a component of the nodule-specific ER signal peptide peptidase complex. In dnf1 mutant, the signal peptide sequences of nodule cell-symbiosome secreted proteins cannot be cleaved, the proteins retain in the ER of nodule cells. The disruption of the nodule cell-symbiosome signaling network leads to defections in symbiosome differentiation and nitrogen fixing symbiosis. Therefore, dnf1 mutant can be used as a novel tool to investigate the proteins function in nodule cell-symbiosome interacting network. To research into the atlas of nodule cell-symbiosome interacting network and its molecular functions, this project will perform researches on 3 aspects: (1) Isolate ER proteins and symbiosome proteins from wild type and dnf1 mutant nodule cells and perform proteomics assay by iTRAQ methods. (2) Find specifically accumulated proteins in WT and dnf1 respectively by bioinformatics analyses, build a sophisticated atlas of nodule cell-symbiosome interacting network. (3) Choose important protein candidates from the network for functional validation, combine genome editing and other methods to study their functions in regulating nodule cell-symbiosome interacting network and symbiosome differentiation. Draw the atlas of nodule cell-symbiosome interacting network and research into its functions can shed light on new biological mechanisms of how host-symbiosome interactions in nodule cells regulate the process of legume-rhizobia symbiosis.