豆科植物与根瘤菌之间的共生固氮系统对农业、生态具有重要意义。延缓根瘤衰老、延长固氮时间，是协调大豆固氮效率与氮肥施用之间关系的有效途径。半胱氨酸蛋白酶在豆科植物中广泛存在并参与调控根瘤的衰老，明确半胱氨酸蛋白酶抑制蛋白在根瘤衰老中的作用及调控机理是发现参与调控根瘤衰老的半胱氨酸蛋白酶的有效方法。本项目前期工作已鉴定出半胱氨酸蛋白酶抑制基因Glyma18g12240,在此基础上，本项目将此基因转化到大豆中，并通过石蜡切片观察、细胞凋亡检测、电镜观察等方法对Glyma18g12240转基因植株中根瘤的衰老特征进行研究；通过酵母双杂交等方法筛选与其互作的半胱氨酸蛋白酶，并验证它们在根瘤衰老中的生物学功能；通过酶活性抑制实验检测Glyma18g12240对其调控的半胱氨酸蛋白酶的活性抑制效应。本项目的开展将为揭示根瘤衰老提供分子生物学证据，对提高大豆品种的固氮效率具有重要的理论价值和应用前景。

The root nodule symbiosis (RNS) between legume plants and rhizobia has critical importance in agriculture and mesology. Delaying the nodule senescence and extending the nitrogen-fixation time are effective ways to coordinate the relationship between nitrogen fixation efficiency and nitrogen application. Cysteine proteases are widely existed in legumes and involved in regulating nodule senescence. Exposing the biological function and regulation mechanism of cystatins in the nodule senescence is an effective method to find the cysteine protease involved in the regulation of nodule senescence. This study use a soybean cystatin Glyma18g12240, which identified from our previous study, as the candidate gene for soybean transformation, by paraffin section observation, TUNEL detection and electron microscopy observation to study the nodule senescence of transgenic plants of Glyma18g12240; by yeast two-hybrid method, and so on, to screen the cysteine proteases, which interacted with Glyma18g12240, and test their biological function in nodule senescence; by cysteine protease activity inhibition assay to detect the inhibitory effect of Glyma18g12240 on the cysteine proteases, which regulated by Glyma18g12240. This study provided the evidence of molecular biology for revealing the nodule senescence, and has important theoretical value and application prospect for improving the efficiency of nitrogen fixation of soybean.