蛋白S-亚硝基化在铝诱导花生细胞程序性死亡中的作用及调节机制

The role and regulatory mechanism of protein S-nitrosylation in Al-induced programmed cell death(PCD) in peanut will be studied with 99-1507(Al-tolerant) and Zhonghua 2(Al-sensitive) as materials. Under Al stress(PCD-induced), Al+NO, Al+cPTIO(nitric oxide scavenger), protein S-nitrosylation levels and mitochondria function changes are detected, S-nitrosylation levels and activities of GSNOR, NADPH oxidase, caspase-like proteases will be analyzed.The types,levels and sites of S-nitrosylation proteins will be analyze by nitrosoproteomic identification. Gene cloning and expression of some main S-nitrosylation proteins are studied such as GSNOR, NADPH oxidase, caspase-like proteases. The results will elucidate the regulatory mechanism of S-nitrosylation on Al-induced PCD. On one hand, SNOs, which are controlled by GSNOR, modify the functions of some proteins related to PCD produce through S-nitrosylation, and regulate Al-induced PCD production in the terminal of signal transduction of mitochondria-dependent PCD mediated by H2O2. On the other hand, NADPH oxidase is activated by Al stress signal through S-nitrosylation elimination, and catalyzes H2O2 production. However, NADPH oxidase is deactivated by nitric oxide through S-nitrosylation, H2O2 signal from Al stress is inhibited, and Al-induced PCD is inhibited.

以花生为材料，研究蛋白S-亚硝基化在铝诱导花生PCD中的作用及机制。进行铝、铝+NO、铝+NO清除剂等处理，分析根尖蛋白S-亚硝基化水平，线粒体功能，GSNOR、NADPH氧化酶、类Caspase等的S-亚硝基化水平和活性，利用蛋白组学技术，鉴定参与铝诱导PCD的S-亚硝基化修饰蛋白种类、水平及位点，对主要S-亚硝基化蛋白（如GSNOR、NADPH氧化酶、类Caspase等）进行基因克隆和表达分析。以阐明S-亚硝基化调节铝诱导PCD发生的可能机制：一是通过GSNOR调节胞内SNOs，对类Caspase等PCD发生相关组分进行亚硝基化修饰，在H2O2介导依赖线粒体的PCD信号通路末端进行调节。二是铝胁迫信号激活NADPH氧化酶去S-亚硝基化，催化产生H2O2；而合适浓度外源NO对NADPH氧化酶进行S -亚硝基化，解除其活性，在信号途径上游调节PCD发生。

蛋白S-亚硝基化是NO发挥作用的重要蛋白翻译后修饰类型。通过研究S-亚硝基化在铝诱导花生根尖细胞程序性死亡中的作用机制，获得主要结果如下：.1.在正常生长的花生根尖中存在丰富的内源性S-亚硝基化蛋白，铝胁迫增强花生根尖蛋白S-亚硝基化水平，但2个品种之间的亚硝基化水平不同。.2．铝敏感品种ZH2的SNOs含量与细胞死亡率、O2.−和H2O2含量呈极显著正相关。外源NO显著提高AhGSNOR基因和蛋白的表达量。.3. 采用ISA法在铝胁迫及对照根尖中鉴定到402个发生S-亚硝基化蛋白，551个修饰位点。单个半胱氨酸位点发生亚硝基化修饰的蛋白占86.8%。铝胁迫诱导128个蛋白的亚硝基化修饰水平显著上调，1个蛋白显著下调。这些差异蛋白主要参与代谢、细胞过程和单组织过程。差异蛋白中有13个与PCD相关。.4. 从花生根尖cDNA中克隆到5个关键亚硝基化蛋白AhGSNOR、AhATPβ、AhPrx51、AhPME2和AhACLA的基因全长，亚硝基化靶位点在线预测5个蛋白均有亚硝基化位点。.5. 构建了5个关键亚硝基化蛋白AhATPβ、AhPrx51、AhPME2、AhACLA和AhGSNOR基因的原核表达载体，经IPTG诱导，均在BL21菌株中高效表达，AhATPβ、AhPrx51和AhPME2重组蛋白以包涵体形式存在，而AhACLA和AhGSNOR蛋白以可溶及包涵体形式存在。.6. 通过镍亲和层析法，分离得到纯度较高的AhGSNOR蛋白，体外实验表明AhGSNOR重组蛋白能够发生亚硝基化修饰，亚硝基化修饰能够引起AhGSNOR活性下降。.7. 通过遗传转化，获得转AhGSNOR和AhMC1正义和转反义基因的烟草植株。转AhGSNOR基因植株具有晚花表型。铝胁迫下，过表达AhGSNOR的烟草植株根系SNOs含量和细胞死亡率显著降低，根相对生长率显著增加，具有更高的根系抗氧化酶活性。表明AhGSNOR基因是铝诱导PCD的负调控因子。.转AhMC1植株叶柄和叶片变短。铝胁迫下，正义植株的类Caspase-3活性、根尖细胞死亡、铝含量、膜脂过氧化程度显著高于野生型，根相对伸长率、SOD、CAT、POD活性显著低于反义和野生型。表明花生AhMC1为铝诱导花生根尖细胞程序性死亡的正调控因子。.最后，提出了NO介导的蛋白S-亚硝基化在铝诱导花生根尖PCD中的作用机制。