类caspase调控铝诱导花生根尖PCD作用机理的研究

This project is to uncover functional mechansim of caspse-like in programme cell death (PCD) induced by aluminum (Al) in the roots of peanut cultivar Zhonghua 2, based on the description of caspase-like in cell death on physiology and biochemistry, molecular biology and morphostructure. The designed strategy mainly includes the obtainment of caspase-like-N that is the most relevant with PCD by characterizing using inhibitors associated with activity of fluorescent substrates and artificate substrates, the construction of vector Ahc-N and RNAi which are introduced into tabacco or peanut, in order to obtain transgenetic plants in which Ahc-N expression is up- or down-regulated, or silenced. Furthermore, the relationship between caspase-like,PCD and senescence-associated genes (SAGs) is determined and analysized using above obtained plants. Then, the location of caspase-like in cell by analysizing the results from the localization of Ahc-N under immnuo-electron microscope and fluorescence microscope during the process of Al-induced PCD. A possible mechanism of caspase-like controlling Al-induced PCD will be certified as follows. Al stress produces ROS burst, ROS activates caspase-like,activates other protease including SAG in turn, then vacuole breaks,cell organelle disintegrates, and produce PCD. The conclusion will provide a new sight to understand the regulation mechansim of caspase-like in PCD and further vertify moleculer mechanism of Al-induced PCD in plants.

以花生品种中花2号为材料，以类caspase在细胞死亡中的作用为核心，通过生理生化、分子生物学和形态结构三个方面研究类caspase在铝诱导花生根尖产生PCD中的作用及其机制。通过荧光底物及人工底物活性抑制剂测定获得与细胞程序性死亡（PCD）相关性最大的类casapse-N;构建Ahc-N、RNAi载体，转入烟草或花生，获取Ahmc-N表达上调、下调或沉默的转基因材料，利用转基因材料进一步分析类caspase、PCD和衰老基因（SAG）之间的关系；通过免疫电镜和荧光显微镜技术检测Ahmc-N在铝诱导PCD发生进程中的亚细胞定位。阐明铝处理导致活性氧迸发，激活类caspase，进而活化SAG等蛋白酶，导致液泡膜破裂，细胞器解体，引发PCD的可能机制。研究结果对明确类caspase在铝诱导PCD中的调控机制，阐明铝诱导PCD机制具有重要科学意义。

铝（Al）毒害严重影响酸性土壤中农作物的产量和质量，对铝毒害的研究已有较多报告，但植物铝毒害和耐性机制仍然不清楚。花生是我国的重要油料作物，而对花生铝毒害和耐性机理的研究还较少。本实验以花生品种99-1507（耐铝型）和中花2号（敏感型）为材料，检测100µmol /L AlCl3处理0、4、8、12h的花生根尖蛋白提取液中类caspase-1-, -2-, -3-, -4-, -5-, -6-, -8-和-9，并在添加特异抑制剂的情况下，比较根伸长生长、根尖细胞死亡率、根尖铝离子含量、苏木精染色、DNA ladder、DAPI、类caspase活性等指标的变化，确定caspase-3-like是Al诱导PCD发生关系最密切（活性最高）的类caspase蛋白酶，并且对敏感型品种的影响高于耐铝型。. 同时利用RACE技术克隆到花生Ahcas-3，其ORF为编码序列1068bp，编码355aa的cDNA序列，通过RT-PCR, west blot等实验，推测Ahcas-3属于诱导增强型基因，其表达诱导或促进PCD的发生，Al诱导的Ahcas-3基因表达与耐铝性负相关，同时促进衰老基因AhSAG表达，且在其上游进行调控作用，促进PCD的发生。. 加入Xho I 和Sal I两个酶切位点及保护碱基，构建Ahcas-3基因的正义、反义植物表达载体，获得转基因植株。比较对照、正义和反义表达的植株中的线粒体的生理变化及亚细胞定位及其与PCD发生的关系，发现铝处理后正义转基因植株根尖积累的铝更多，膜脂过氧化水平更强，活性氧通过引起线粒体膜脂过氧化，改变线粒体膜通透性，激活提高类caspase-3的活性，诱导PCD的发生。