GmGH3.1a与结瘤因子受体互作调控大豆共生固氮的分子机制研究

Nitrogen is a main factor limiting crop production. Soybean can obtain nitrogen through symbiosis with a nitrogen-fixing bacterial microsymbiont (broadly called rhizobia). Nodulation can be divided into rhizobia infection and nodule development. Up to now, few genes has been identified in regulating the efficiency of symbiotic nitrogen fixation. Our earlier study showed that overexpression of GmGH3.1a dramatically promoted root hairs deformation upon rhizobial infection and increased the number of nodules. Most importantly, we found that GmGH3.1a physically interacts with Nod factor (NF) receptor GmNFR1α. In this study, we aim to 1) confirm the genetic relationship between GmGH3.1a and GmNFR1α; 2) uncover the biochemical mechanism underlying GmGH3.1a-GmNFR1α interaction and investigate how GmGH3.1a-GmNFR1α interaction affects GmGH3.1a stability and enzymatic activity; 3) analyze the role of auxin homeostasis mediated by GmGH3.1a phosphorylation in early infection process, nodule formation and symbiotic nitrogen fixation of soybean, as well as plant sensitivity to rhizobia; 4) identify the natural variations of GmGH3.1a that confer enhanced SNF efficiency. The results will uncover the mechanism by which GmNFR1α regulates rhizobial infection and the efficiency of SNF through directly targeting GmGH3.1a and modulating auxin homeostasis. The goal is to establish the direct link between NF signaling and auxin, and to elucidate the molecular mechanism through which NF signal is transduced to modulate auxin-mediated soybean nodulation.

大豆通过共生固氮获取氮素，保证其产量和品质。目前大豆共生固氮调控机制了解甚少。本项目以编码生长素螯合酶的GmGH3.1a为研究对象，前期发现GmGH3.1a过表达稳定转化植株表现根瘤数明显增加，叶片叶绿素含量升高。初步证明GmGH3.1a与结瘤因子（NF）信号通路的受体激酶GmNFR1α互作，预示GmNFR1α可能直接将NF信号与调控生长素稳态整合。本研究中，我们将：1）明确GmGH3.1a与GmNFR1α调控根瘤发育时的互作及遗传关系；2）探明其中的生化机制及GmGH3.1a被磷酸化对其生长素螯合酶功能的调控作用；3）GmGH3.1a介导的生长素恒稳态在早期侵染及共生固氮过程中的作用；4）挖掘GmGH3.1a的优异等位变异。研究结果将将首次揭示植物感受NF后直接调控生长素恒稳态介导结瘤早期过程和共生固氮效率的分子机制，将为利用标记辅助选择和分子设计育种提高大豆的共生固氮效率提供数据支持。

共生固氮是植物获取氮素的重要生物学过程，其中早期菌植互作对共生固氮具有决定性作用。项目申请时基于GmGH3.1a与GmNFR1α可能存在蛋白水平互作的相关信息，拟研究内容包括：（1）系统研究GmGH3.1a的功能及其与GmNFR1α的互作关系；（2）GmGH3.1a与GmNFR1α互作的生化机制研究；（3）GmGH3.1a介导的生长素恒稳态在早期侵染及根瘤形成和共生固氮过程中的作用；（4）挖掘GmGH3.1a的优异等位变异。项目批复后，进化树分析确认候选蛋白为生长素螯合酶GH3.1的同源蛋白GmGH3.1b。功能研究证实GmGH3.1b在大豆根瘤的发生发育过程中发挥重要的调控作用。经过2019年度在GmGH3.1b与结瘤因子受体NFR1α互作及磷酸化方面的实验验证，显示二者可能不存在体内的互作及磷酸化。但相关结果证明GmGH3.1b与负向调控大豆结瘤的异源三聚体G蛋白亚基GmGα4存在互作，之后利用多体系证明GmGH3.1b与异源三聚体G蛋白亚基GmGα4的互作关系。在双分子荧光互补技术（BiFC）和萤火虫荧光素酶互补成像法（LCI）体系中证明GmGH3.1b也可与此前已被证明可以正向调控大豆的结瘤过GmGβ4和GmGγ3互作。该结果暗示GmGH3.1b可能会在异源三聚体G蛋白的解离及调控大豆结瘤过程中发挥作用。除此之外，基于GmGH3.1b在接菌早期对根瘤菌侵染的显著响应，近期我们也在开展其转录调控机制的研究工作，重点是鉴定和解析转录因子对GmGH3.1b的转录激活机制。. 综上所述，项目基金的资助对GmGH3.1b调控大豆响应根瘤菌侵染及结瘤固氮的分子机制研究提供了重要帮助。取得的相关结果为进一步解析GmGH3.1b介导大豆早期菌植互作关系建立过程的分子调控机制提供了更多的数据支持。