【作者】 齐耀程；

【导师】 张炜；

【作者基本信息】 南京农业大学 ， 生物化学与分子生物学， 2010， 博士

【摘要】 细胞程序性死亡(PCD)在植物的发育、抗病及植物与环境互作等过程中发挥着极其重要的作用。而线粒体作为细胞重要的细胞器,在植物PCD过程中具有关键性调节功能。本实验室前期研究发现,水稻幼苗在500mM NaCl胁迫下,其根尖细胞出现典型细胞程序性死亡特征。根尖细胞的线粒体蛋白质组分析也表明线粒体热激蛋白70(mtHSP70)在PCD早期的表达量显著上升。为了研究mtHSP70在PCD中发挥的生物学功能,本研究以日本晴水稻悬浮细胞系为受体材料,在水稻悬浮细胞中导入未经改造的pCAMBIA1304载体的作为空载体对照(VC),过表达mtHSP70(?)悬浮细胞(OX)为材料制备原生质体,48℃水浴热激处理15min后发现,在28℃恢复培养条件下水稻原生质体随时间延长出现明显的PCD特征,包括DNA片段化、细胞核呈TUNEL阳性、染色质凝集、线粒体膜电势显著降低等。VC原生质体在热处理后,恢复培养2h其基因组DNA可降解出现明显的DNA Ladder,而OX原生质体DNA Ladder出现于恢复培养4h左右。100mM H2O2引发原生质体DNA Ladder (?)勺结果与热诱导相似。热诱导下VC原生质体死亡率6h可达到63.2%,OX仅为39.3%。H2O2处理时VC原生质体死亡率6h即可达到91%,OX也仅为43%。将热处理的原生质体利用Hoechst33342荧光染色观察到热激后细胞核破裂和染色质凝聚等PCD形态学变化,VC细胞核裂解程度较大,OX原生质体恢复培养6h的核裂解程度相当于VC恢复培养12h的状态；经TUNEL染色可以发现,虽然两类原生质体进入PCD的细胞数目随时间延长而都呈增长趋势,但是OX恢复培养24h的阳性率为70%,而VC原生质体的阳性率却已超过90%,这说明过表达mtHSP70可以降低Ca2+或Mg2+依赖性核酸内切酶的活性,延缓了PCD的发生进程；热激处理均可诱导两种类型原生质体的细胞色素c从线粒体释放到细胞质中,Western Blot检测显示细胞色素c释放量15min内随热处理时间延长而增大。热激诱导PCD初期,VC原生质体内活性氧爆发并于1.5h和2.5h出现两个爆发峰,而OX原生质体内活性氧的水平稳步上升,避免了短时间内突发的极端氧化伤害；线粒体膜电势测定发现热激诱导的原生质体PCD过程中,线粒体膜电势均有下降,但VC原生质体中线粒体膜电势下降速度较快,表明过表达mtHSP70细胞的线粒体膜完整性高,线粒体损伤程度小；VC原生质体的类Caspase-3活性随恢复培养时间延长而增强,其6h的相对荧光值是OX的8倍,而OX的荧光强度一直保持平稳的本底水平,可能是原生质体通过过表达mtHSP70稳定线粒体膜电势防止促PCD因子的释放而抑制了胞质内类Caspase-3的活化。本研究证明了线粒体热激蛋白70通过稳定线粒体膜电势和抑制活性氧爆发,有效延缓了热激诱导下水稻悬浮细胞原生质体PCD的发生。更多还原

【Abstract】 Programmed cell death (PCD) plays an important role on plant growth and development, disease resistance and the interactions between plant and its environment. Mitochondrion, as one of the key organelles, regulates the main progress of PCD. The preview study established a salt stress-induced PCD model in rice root tip cells, while mtHSP70was up-regulated more than2-fold in mitochondrial proteome after salt stress. In this study, we found that heat shock treatment resulted in some typic PCD progresses in rice protoplasts. A series of relevant studies had carried out to investigate the functions of mtHSP70in PCD. The vector control (VC) and over-expression (OX) mtHSP70rice protoplasts from different suspension cell lines were researched under the treatment of heat shock at48℃for15min and returned to the normal conditon, the both types of protoplasts showed the specific PCD features, such as DNA Ladder, nuclear condense and deformation, TUNEL positive reaction and mitochondrial membrane potential decrease. It was proved that the chromatin condensation, nuclei deformation and genome DNA would degradated under heat shock via Hoechst33342, but the chromatin condensation in OX protoplasts was similar to VC at6h. It was found that the number of TUNEL positive nuclei of OX protoplasts was less than VC under the heat shock treatment, the positive rates of VC and OX were90%and70%, indicating that the activities of Ca2+/Mg2+-dependent endonuclease were decreased. Results indicated that, comparing to VC protoplasts, OX were resistant to PCD in many fields. For OX protoplasts, the thermo treatment could induce the appearance of DNA Ladder at4h, while appeared at2h in VC. Death rate test under H2O2treatment revealed the similar tendency to heat shock treatment. The death rate of heat shock in VC approached63.2%at6h, but OX was39.3%, while100mM H2O2treatment made the death rates of VC and OX rising to91%and43%respectively. Cytochrome c released from mitochondria could be detected immediately after heat shock; the optical density of VC was calculated more than1.0, while OX showed less than0.7.H2DCFDA staining suggested that OX protoplasts could avoid the injuries of the oxidative burst as the ROS level remained stability and up-regulated slowly, while the VC protoplasts occurrent bimodal in prophase. The specific probe JC-1displayed that mitochondrial membrane potential decreased fast when heat shock from5min to15min, and OX protoplasts were slower than VC, indicating, attributed to the over expression of mtHSP70, the excellent integrity and low mitochondrial membrane injuries of the OX protoplasts. The Caspase-3-like was specifically activated after heat shock, OX protoplasts showed significantly less Caspase-3-like activity than the VC. The RFU of VC protoplasts was more than8-fold to OX. It indicated mtHSP70over expression may inactivate Caspase-3-like by maintaining the mitochondrial membrane potential.All the results above suggested that mtHSP70may delay the plant PCD by maintaining mitochondrial membrane potential and inhibiting cellular ROS bursts in rice suspenssion cells.更多还原