【作者】 李云霞；

【导师】 韩烈保；

【作者基本信息】 北京林业大学 ， 草业科学， 2009， 硕士

【摘要】 结缕草是一种品质良好的草坪草,但随着外界环境的日益恶化,其生存环境也受到了破坏。环境胁迫对植物细胞的伤害在很大程度上是由于活性氧浓度上升所致,活性氧已成为环境胁迫诱导细胞程序性死亡的一个重要信号分子。本研究以日本结缕草（Zoysia japonica Steud）悬浮细胞为试验材料,通过外源添加活性氧H₂O₂,探讨H₂O₂胁迫下的结缕草细胞死亡是否为程序性死亡,此外,在H₂O₂胁迫下外施GA3,探讨GA3在此过程中的作用,为研究结缕草抗逆机理奠定理论和实践基础。主要结果如下:（1）用不同浓度H₂O₂胁迫处理,通过细胞密实体积体积法,测定H₂O₂胁迫下细胞的生长状况,发现H₂O₂胁迫抑制细胞生长,并于胁迫2天后停止生长。伊文思蓝染色检测细胞死亡动态,结果表明结缕草细胞会随着H₂O₂处理浓度和培养时间的增加而加剧死亡。（2）显微观察结果:台盼蓝（Trypan Blue）染色表明,低浓度（5mmol/L）H₂O₂胁迫不会使细胞膜结构破坏、细胞着色,随着H₂O₂浓度加大,30mmol/L H₂O₂胁迫会使细胞膜结构破坏、细胞着色;DAPI荧光染色结果表明,在H₂O₂诱导结缕草悬浮细胞发生的死亡过程中出现了原生质浓缩、染色质凝集、细胞核皱缩、凋亡小体等细胞程序性死亡的形态特征。DNA含量分析结果表明,H₂O₂胁迫12h后,细胞DNA含量下降,24h后,DNA大量降解。（3）H₂O₂诱导结缕草细胞程序性死亡过程中,SOD、POD、CAT等抗氧化酶活性有不同程度下降,表明它们在活性氧代谢中可能发挥不同作用。当诱导24h后,抗氧化酶活性的变化开始趋于平缓。（4）在H₂O₂胁迫下,于悬浮细胞外施GA3后,DAB染色表明氧化胁迫主要是在胁迫初期发生,而GA3的添加可减少活性氧产生。细胞死亡动态检测结果表明,用10mmol/L GA3处理细胞,对细胞生长状态影响不太,细胞死亡率低;在H₂O₂中添加GA3后,与只添加H₂O₂诱导的细胞死亡数量相比,减少了细胞死亡量。添加GA3能增强SOD、POD、CAT活性,表明GA3对H₂O₂诱导的结缕草细胞程序性死亡有部分抑制作用。更多还原

【Abstract】 Zoysiagrass（Zoysia japonica Steud.） is a type of turfgrass with good quality,however,as the deteriorating external environment,the living environment which they live had been destroyed.Environmental stress on plant cell damage was largely due to the increased concentration of reactive oxyen species,reactive oxyen species had become an important signal molecule which induced programmed cell death by environmental stress.Japanese lawngrass（Zoysia japonica Steud.） cells were uesd as material,through added exogennous reactive oxyen species-H₂O₂ to investigate whether the cell death under H₂O₂ stress was programmed cell death.In additon,added exogenous GA3 into H₂O₂ to explore the role of GA3 in the process,in order to provide theoretical and practical basis for studying zoysiagrass resistance mechanism. Conclusions showed as follows:（1）Through the adding of different concentrations of H₂O₂,the cell growth coditions were determined by packed cell volume.H₂O₂ sress could inhibit the growth of the cells and japanese lawngrass cells could no longer grow after 2 days of H₂O₂ sress.Evans blue staining detected cell death dynamics,the results showed the nummber of cell death would increased with higher H₂O₂ concentration and longer treatment time.（2） The results of microscopic observation:trypan blue staining indicated that low level of H₂O₂ （5mmol/L） would not damaged the membrance strcture,,and cells would not stained.With higher concentration,cells stained under 30mmol/L H₂O₂,the membrance strcture were damaged.The results of DAPI stained nuclei indicated there were morphological characteristics of programmed cell death in the process of japanese lawngrass cell death inducd by H₂O₂, such as protoplast shrinkaged,chromation condensed,nuclei shringkaged,and had some apoptosis bodies.DNA content analysis displayed that cell death was increased after 12 hours H₂O₂ disposal,a large number of DNA degraded after 24 hours.（3） Antioxidant enzyme activity as SOD、POD、CAT decreased in the process of cell death induced by H₂O₂ demonstrated that they might play different role in the process of activity oxyen metabolism..Antioxidant enzyme activity trends to smooth after 24 hours.（4） After exogenous application of GA3,the results of DAB staining showed that oxidative damage was initiated in the early stage of stress,and released less amount of activity oxyen.The results of cell death dynamics indicated there were little effct on cell growth and low cell death rate treating with 10mmol/L GA3.After added GA3 into H₂O₂,decreased the number of cell death.After exogenous application of GA3,enhanced activity of SOD、POD、CAT indicated that GA3 play an important role in alleviating cells death inducing by H₂O2 in Japanese lawngrass.更多还原