低磷胁迫调控大豆硝酸还原酶GmNR4磷酸化及其氮同化的机制

It is very important to synergistically improve utilization efficiency of both nitrogen (N) and phosphorus (P) fertilizers in agriculture development. It has been documented the physiological and molecular mechanisms underlying synergistic enhancement of both N and P efficiency in crops. However, it remains largely unknown that synergistic enhancement of both N and P efficiency might be achieved through controlling phosphorylation of proteins functions in N assimilation under low phosphorus conditions. Previously, we found that P deficiency led to increased phosphorylation of nitrate reductase 4 (GmNR4) in soybean roots. Furthermore, its expression levels were also increased by P deficiency, strongly suggesting that P deficiency might regulate nitrate assimilation through mediating GmNR4 transcription and phosphorylation levels in soybean. In the present study, we intend to investigate enzymatic activities and stability of GmNR4 and its S491 phosphorylated mutants. Subsequently, growth, N and P content will be further analyzed in transgenic soybean hairy roots and soybean whole plants with suppression of GmNR4, overexpression of GmNR4 and its S491 phosphorylated mutants. Meanwhile, yeast hybridization and bimolecular fluorescence complementation (BiFC) will be conducted to identify the protein kinase interacted with GmNR4. All the results together will elucidate adaptive strategies of soybean to P deficiency through controlling GmNR4 phosphorylation, which will be helpful to provide the theoretic basis and gene germplasms for developing soybean varieties with high P and N efficiency.

如何提高作物氮磷肥的协同高效利用是农业生产的重要问题。以往的研究表明，作物存在氮磷协同高效的生理和分子机制。但是，关于低磷胁迫是否通过调控参与氮同化蛋白的磷酸化水平，以达到作物氮磷协同的机制鲜有报道。我们前期通过比较磷酸化蛋白谱的方法，在大豆根系中发现低磷胁迫显著上调硝酸还原酶4(GmNR4)的磷酸化水平。而且，其基因表达量也显著受低磷胁迫的上调，暗示了低磷胁迫可能在基因转录水平和蛋白磷酸化水平共同参与调控大豆对硝酸盐的同化。本研究拟在比较GmNR4及其S491磷酸化点突变的蛋白酶活及其稳定性的基础上，进一步分析抑制GmNR4、超量GmNR4及其点突变表达对大豆毛根和整株生长及其氮磷吸收的影响。同时，结合酵母双杂交和双分子荧光技术，初步明确与GmNR4互作的蛋白激酶。以期明确GmNR4磷酸化水平修饰是大豆根系响应低磷胁迫的适应性机制，为选育氮磷协同高效的大豆品种奠定理论基础和候选基因。

本项目以明确在大豆根系中对低磷胁迫响应的GmNR4的功能研究为主线，开展了相关的植物生理和分子生物学的研究，主要获得的研究成果如下：1）通过磷酸化点突变、亚细胞定位及酶活性分析等，明确了硝酸还原酶 GmNR4 的 S491 位点的磷酸化修饰抑制了该蛋白的酶活性，从而调控大豆根系在低磷条件下对硝酸盐的同化作用和生长；2）对超量和敲除GmNR4表达的、稳定遗传的大豆离体毛根转基因后代进行分析，发现大豆离体毛根对磷和氮的吸收能力受GmNR4表达水平的调控；3）以GmNR4-BD为诱饵，通过酵母cDNA文库的筛选及酵母双杂实验，获得了与GmNR4互作的蛋白激酶GmFAB1C-1，并通过初步的功能验证，明确了蛋白激酶GmFAB1C-1通过调控GmNR4的磷酸化水平，控制了磷胁迫条件下大豆根系的氮同化作用，达到大豆的氮磷协同高效。总之，本项目通过以上的研究，初步解析了低磷胁迫通过调控 GmNR4 的磷酸化水平控制大豆根系硝酸盐同化的分子机理。该研究结果为丰富豆科作物氮磷协同高效的机制奠定基础，为作物氮磷协同高效的遗传改良工作提供候选基因。