水稻生殖发育的信号网络与分子机制已成为水稻研究领域的热点问题之一。本实验室前期研究结果表明，水稻花粉特异表达的钙依赖性蛋白激酶家族成员OsCDPK21参与调控水稻花粉发育过程：21-RNAi转基因水稻的花粉在空泡期之后接近成熟期时出现了凋亡，最终导致水稻花粉败育。在植物细胞中，活性氧（ROS）被认为是引起细胞凋亡的重要因素。ROS是一类具有氧化能力的分子、离子和自由基，主要包括超氧阴离子、过氧化氢、羟自由基等，它们广泛存在于机体内并参与调节机体重要的生理及病理过程。许多研究表明，ROS通过对生物膜的损伤、蛋白质的氧化损伤、DNA的氧化损伤、影响信号转导和基因表达诱发细胞凋亡。植物细胞内ROS是由 respiratory burst oxidase homolog (rboh) 蛋白产生的。在拟南芥基因组中共发现10个同源基因（AtrbohA–J），参与细胞应激反应、细胞生长、激素响应、气孔关闭、免疫应答反应、细胞凋亡等多个生理过程。在水稻中，至少发现9个rboh基因。基于以上研究结果，我们提出假设一：OsRbohs参与了由OsCDPK21基因沉默引起的花粉败育。实验室前期通过酵母双杂交实验并未筛选到与OsCDPK21直接互作的OsRboh家族蛋白；但筛选到一个14-3-3蛋白。该蛋白是典型的接头蛋白，即作为接头或者支架将两个蛋白质联系在一起使其相互作用。基于该结果，我们提出假设二：OsRboh蛋白与OsCDPK21之间的相互作用是由14-3-3蛋白介导的。为确定OsCDPK21与OsRboh蛋白是否具有互作关系以及OsCDPK21是否影响OsRboh蛋白活性，本实验构建了OsCDPK21、OsRboh以及14-3-3蛋白的动物表达载体和原核表达载体，在HEK293T细胞中用western-blot方法验证了三个植物基因在动物细胞当中的表达，并对三者互作关系进行了研究,对OsRboh蛋白活性进行了测定。获得了如下结果：1、本研究构建了OsCDPK21、OsRboh以及14-3-3蛋白原核表达载体，并获得可溶性蛋白。2、本研究构建了OsCDPK21、OsRboh以及14-3-3动物表达载体，通过western-blot方法验证，确定在HEK293T细胞中三个植物基因在动物细胞中成功表达。3、在瞬时转化OsRboh动物表达载体的HEK293T细胞中，测定了ROS浓度，确定了动物细胞OsRboh活性检测体系。4、构建了OsCDPK21、OsRboh以及14-3-3连有GFP的动物表达载体，研究了三者在HEK293T细胞的亚细胞定位。结果表明OsCDPK21、OsRboh定位在质膜上，而14-3-3在整个细胞当中分布。

Rice reproductive development network and the signal mechanism has become a major subject in rice research. It is well known that calcium is a key regulator of pollen development, pollen tube growth and flowering, and calcium-dependent protein kinase (CDPK) is an important receptor of Ca2+ signal. Previous studies proved that OsCDPK21 play an important role in pollen development. CDPK21-RNAi transgenic lines show that the development of rice pollen was blocked after vacuole stage, which eventually led to the loss of rice pollen and was considered as programed cell death (PCD).Reactive oxygen species (ROS) have been proposed as key inducers for PCD. ROS may cause oxidative damages of DNA, lipids, and proteins and may impair the cellular functions. More importantly, some studies demonstrated that these ROS at moderate concentrations may also act as signal transduction messengers and modulate gene expression. In short ROS play multifaceted signaling functions mediating the establishment of multiple responses and can act as local toxins. In plant, ROS is generated by respiratory burst oxidase homolog (rboh). Based on the above, there is an interesting suppose maybe OsRbohs involve in CDPK21-RNAi induced rice pollen deficiency. Furthermore, previous studies show 14-3-3 direct interacts with OsCDPK21by Yeast double hybrid assay, and 14-3-3 protein reveals the lever protein character. Easy to think it is probably the interaction of OsRboh and OsCDPK21 be mediated by 14-3-3 protein.In order to investigate the interaction between OsRboh and OsCDPK21, and detect the activity of OsRboh, we studyed OsRbohs in a heterologous expression system using HEK293T cells and successfully had the expression and localization of OsCDPK21、OsRboh、14-3-3 in HEK293T cells.