一氧化氮调控铝诱导花生根尖细胞程序性死亡机理的研究

With 99-1507 (Al-tolerant) and Zhonghua NO.2 (Al-sensitive) as materials, from morphologic structure,physiological and biochemcial changes, and molecular biology aspects, the mechanism of nitric oxide (NO) inhibiting Al-inudced programmed cell death (PCD) in peanut root tips will be studied. Under Al stress, Al+SNP, Al+different inhibitors, some physiological and biochemical index are detected including cell dye,the contents of Al, NO,SNO,cGMP,and Ca2+, DNA ladder, the activities of antioxidant enzyme, GSNOR,MBP,NOS,NR and Caspase 3, transcriptomics，gene expression profile,and differentially expressed genes and metabolic pathway of NO controlling Al-induced PCD. The genes of MAPK and GSNOR will be cloned, the temporal and spatial expression patterns will be detected. The results can elucidate the mechanism of NO inhibiting Al-induced PCD. Firstly, NO can decrease the ROS burst by Al stress as ROS scavenger, Al adsorbent rate and play direct effect. Secondly, as a signal factor, NO can control gene expression and play indirect effect by signal pathway of cGMP and MAPK. The results can improve the knowledge of the effects of NO in plant growth and development, elucidate the mechanism of Al-induced PCD in peanut root tips.

以花生品种99-1507（耐铝）和中花2号（铝敏感）为材料，从形态结构、生理生化和分子生物学三个方面研究NO抑制铝诱导花生根尖细胞PCD作用机制。通过利用铝胁迫、铝胁迫＋SNP、铝胁迫＋抑制剂处理，检测细胞染色、铝、DNA梯、ROS、NO/SNO、cGMP、Ca2+、抗氧化酶、GSNOR、MBP、NOS/NR、Caspase3活性等变化，进行转录组学和表达谱分析，寻找铝诱导PCD下NO调控差异基因及其控制代谢途径，进行MAPK和GSNOR等基因全长克隆，检测其时空表达。以证实NO抑制铝诱导PCD发生的可能机制：一是作为ROS的清除剂，减轻铝胁迫引起迸发，降低铝吸附率，而直接发挥作用；二是作为信号因子，通过cGMP、MAPK等信号转导途径，调控有关基因表达，发挥间接效应。研究结果对丰富NO在植物生长发育过程中作用的认识，阐明铝诱导PCD机制，具有重要意义。

铝是酸性土壤作物生长的主要限制因子，但植物铝毒害和耐性机制尚不明确。我们在发现铝诱导花生根尖细胞程序性死亡（PCD）、PCD发生与耐铝性负相关、一氧化氮（NO）缓解铝诱导PCD的基础上，研究了NO调控铝诱导PCD机理。主要结果如下：.1、建立了验证PCD现象的系统方法，包括DAPI和Evans blue染色、DNA梯、Hrs203j表达、类caspase-3酶活性等。.2、证实铝诱导花生根尖活性氧迸发，抗氧化能力差异是花生品种耐铝性不同的重要原因之一。.3、发现铝诱导花生根尖快速发生PCD，提出铝诱导PCD发生为H2O2介导的依赖线粒体途径。.4、NO对铝诱导PCD起负调控作用，但NO并非作为ROS清除剂而直接调节，而是作为信号分子，通过信号转导，调节H2O2介导的线粒体途径而调控铝诱导PCD的。.5、花生根尖细胞存在G蛋白，cGMP与铝诱导PCD负相关，NO对铝诱导PCD的抑制作用是通过激活cGMP实现的。.6、完成了对照和铝胁迫12h的中花2号花生根尖RNA转录组高通量测序。获得310910个转录本信息，总数据量超过16G，搭建了花生转录组信息平台。获得铝胁迫差异基因43469条，表达上调的有23982个，表达下调的有19487个。发现20个unigenes与NO通路有关，主要有液泡膜铁转运蛋白、NO合成相关蛋白等。.7、克隆到花生部分MAPK成员AhMEKK2、AhMKK4、AhMKK5等，随铝胁迫时间延长，这些基因表达呈现先升高后下降的趋势，处理4h时最高；NO提高它们的表达水平。证实花生中存在MAPK途径，提出与MAPK途径相关的NO抑制铝诱导花生根尖PCD的分子机制，即NO激活MEKK2－MKK4/5—MPK3/6—WRKY33级联反应，上调AhBI-1表达，下调AhPDCD5表达，依赖线粒体途径，抑制PCD发生。.8、克隆到花生AhGSNOR，铝胁迫提高AhGSNOR表达和活性，增加SNOs含量，通过S-亚硝基化修饰AhGSNOR、AhGAPDH，诱导PCD发生。NO提高GSNOR活性，降低SNO含量，通过S-亚硝酰化修饰下调AhGAPDH表达，从而抑制PCD发生。.综合分析，NO调节铝诱导PCD的途径之一为：NO→AhGSNOR等→SNOs→cGMP→H2O2→MAPK→依赖线粒体PCD途径。但SNOs如何调节cGMP等还需深入研究。