玉米/大豆套作调控根瘤固氮的生理生化机制研究

It is well know that nodule nitrogen fixation is an effective way for crops to utilize the nitrogen fertilizer in legume-cereal intercropping system. However, the mechanisms involve physiology and biochemistry are still unclear. Previously, we found the nitrogen utilization efficiency was significant increase in maize-soybean relay intercropping. Together, a important factors which impacts nitrogen fixation is the quality and nodule nitrogen fixation time of nodule. Here, we will use methods including environmental monitoring, enzyme activity detection and quantitative polymerase chain reaction (q-PCR) to research nodule nitrogen fixation under the different root interaction strength of maize and soybean. We will to test the root exudates component and the rhizobium diversity, and to define the parameters of nodules physiological and morphology. In addition,we are also to determine the reactive nitrogen specis(RNS), reactive oxygen species(ROS), NO scavenger (flavohaemolobin), superoxide dismutase (SOD) , catalase (CAT) and activity and dynamic variations of encoding of proteolytic enzymes in this research. Through the research, we hope to clarify the relationships between the soil environment and nodule senescence, and root exudates and nodule formation, and reveal the physiology and biochemistry mechanism involves nodule nitrogen fixationthe in maize-soybean relay intercopping system as well as offers opportunities for further utilize nitrogen efficiently. In addition, this study will provide theoretical basis to technical development for reduction of excessive nitrogen applications in agriculture.

豆科与禾本科间套作是提高资源利用率与生物固氮的有效途径，但其豆科固氮的生理生化机制尚不明确。本研究针对玉米/大豆套作的养分高效利用问题，先前研究发现，玉米/大豆套作可有效提高作物氮肥利用率与根瘤固氮能力，根瘤质量与固氮时间是影响其高效固氮的重要因素。在此基础上，本研究拟通过环境监测、酶活检测和qPCR等方法，对根系分泌物组分与根瘤菌多样性差异、根瘤形态结构与固氮生理参数、根区土壤环境与理化性质的动态变化进行深入研究，检测根瘤中活性氮与活性氧的含量以及NO清除剂（黄素血红蛋白）、抗氧化酶（SOD、CAT）与蛋白质降解酶（半胱氨酸蛋白酶）的含量、活性及其编码基因的表达差异，进一步阐明玉米/大豆套作下大豆根区土壤环境、根系分泌物变化与根瘤形成及衰老的关系，揭示根瘤高效固氮的生理生化机制。这将有助于完善禾豆间套作的氮高效利用理论，为间套作减肥增效技术研发提供理论依据。

本研究以玉米/大豆套作系统为研究对象，结合四年长期定位实验，通过环境监测、酶活检测和qPCR等方法，研究根区土壤通气环境、根系分泌物组分与根瘤菌多样性差异、根瘤形态结构与固氮生理参数的动态变化，检测根瘤中活性氮与活性氧的含量以及抗氧化酶活性及其编码基因的表达差异，阐明套作环境下大豆根瘤形成及延衰机制，为完善禾本科/豆科间套作系统减肥增效技术研发提供理论依据。.（1）玉米大豆套作改善了根区土壤通气环境、根系分泌特性及特异菌群多样性。提高大粒径(>5mm)土壤水稳性团聚体比率31.34%，增加土壤孔隙度1.33%、氧气含量1.73%和呼吸量54.52%，降低土壤容重1.68%。套作增强大豆黄酮分泌，共生期大豆黄素、染料木素分泌量提高85.0%和43.9%；但茉莉酸合成途径关键基因LOX10的表达量下调77.8%，其分泌量降低58.4%。套作降低大豆根际土壤的细菌多样性，不同大豆品种间的根瘤慢生型根瘤属的表达存在差异；除南豆12外，桂夏3号和nts1007在套作时的慢生根瘤菌属均呈下降趋势。.（2）玉米大豆套作改善了根瘤生长发育性状，提高了固氮能力。共生期大豆单株根瘤数量降低，但提高单个根瘤干重；恢复期套作根瘤直径(>0.2cm)显著大于单作。浇灌玉米根系分泌物，促进大豆黄酮合成基因GmIFS1及根系根瘤发育基因GmEXPB2的上调表达，显著提高南豆12单个根瘤干重及根系的总根长、根表面积、根体积及根干重；nifH上调表达1.07倍，提高根瘤固氮酶活性2.22倍。.（3）玉米大豆套作增强了根瘤抗衰老能力。套作提高根瘤的蔗糖含量，施氮有利于前期的蔗糖积累，但大量施氮会降低后期蔗糖含量，降低淀粉积累。通过增加后期抗氧化物酶基因（GmCAT4、GmSOD2）的表达及酶活性（CAT、SOD），提高抗氧化物质（GSH、AsA）的积累，适量施氮实现上下协同作用缓解根瘤蛋白质降解（GmCP2、GmCP15a），延迟NO升高，延缓根瘤衰老。