紫云英二磷酸核苷磷酸酯酶AsPPD1在共生固氮中的作用机制研究

It is known that phosphorus regulates symbiotic nitrogen fixation (SNF). In previous work, we identified a Diphosphonucleotide phosphatase from Astragalus sinicus, AsPPD1, which was associated with cellular phosphorus remobilization and played important roles in SNF. This project aims to reveal its mechanism during the processes of nodule formation and nitrogen fixation by symbiosomes. The project will be carried out via the following three approaches. Firstly, the localization of AsPPD1 to symbiosome membrane will be confirmed by immunoelectron microscopy and western blot techniques. The relationship of AsPPD1 with energy metabolism on symbiosome membrane will be analyzed through measuring the changes of PPD activity, ATP/ADP ratio, energy charge and phosphorus content on symbiosome membrane and in symbisome after AsPPD1 silencing. Secondly, phenotypic recovery was analyzed through supplement of exogenous ATP、ADP or AMP. Thirdly, proteins interacting with AsPPD1 will be screened and identified by yeast two-hybrid technique. The in vito/vivo protein-protein interaction will be verified by using pull-down, bimolecular fluorescence complementation and co-immunoprecipitation techniques. Furthermore, the functions of the target proteins in SNF will be investigated as well. The results of this study will help us to deeply understand how phosphorus regulates SNF. On the other hand, this study is able to provide theoretical basis and novel ideals for genetic improvement of leguminous crops.

磷对共生固氮有调节作用，我们前期研究发现紫云英二磷酸核苷磷酸酯酶AsPPD1可能与磷的循环利用相关，在根瘤形成和共生体固氮中发挥重要作用。为揭示其作用机制，本申请拟从以下3方面展开研究：（1）采用免疫电镜技术和共生体膜蛋白的Western Blot技术确证AsPPD1在共生体膜上的定位；检测AsPPD1沉默后，共生体和共生体膜上PPD酶活、ATP与ADP之比、能荷及磷含量的变化，分析AsPPD1与共生体能量代谢的关系。（2）外源补充ATP、ADP或AMP，分析超表达和RNAi发根的表型恢复情况。（3）利用酵母双杂交技术，筛选鉴定根和根瘤中与AsPPD1互作的蛋白，利用Pull-down、BiFC、Co-IP等技术验证蛋白互作，并研究互作蛋白在共生固氮过程中的功能。研究结果将有助于我们更深刻地理解磷素营养对共生固氮调节的分子机理，同时也可为豆科作物的遗传改良提供理论依据和新思路。

为了研究紫云英二磷酸核苷磷酸酯酶AsPPD1在共生固氮过程中的作用机制，本项目基于前期工作基础，确证了AsPPD1 在共生体上的定位，研究了其表达变化对根瘤ATP/ADP的比值和能荷的影响，筛选到了其可能的互作蛋白，并研究了互作蛋白在共生固氮中的功能。结果表明：(1) AsPPD1定位于共生体膜，且在RNAi根瘤中ATP/ADP的比值和能荷明显降低。（2）利用酵母双杂交文库筛选并验证了2个AsPPD1可能的互作蛋白，MsrB和Cdc25-like，利用BiFC确定了MsrB和AsPPD1互作的结构域。（3）时空表达情况检测结果提示MsrB和Cdc25-like可能与根瘤固氮相关。（4）免疫荧光和免疫电镜结果表明MsrB在根瘤中可能定位于共生体。（5）MsrB超表达对共生表型影响不明显，而RNAi导致根瘤数量减少，发育迟缓，说明AsMsrB的沉默抑制结瘤，同时抑制根瘤的发育。本项目的研究结果为深入研究AsPPD1和AsMsrB在共生固氮中的互作机制奠定基础。