【作者】 杨宗灿；

【导师】 宋纯鹏；

【作者基本信息】 河南大学 ， 细胞生物学， 2011， 硕士

【摘要】 逆境胁迫可以分为非生物胁迫与生物胁迫两大类,严重影响着植物的正常生长发育,其中许多非生物胁迫都可以诱导植物体内产生大量的活性氧（Reactive Oxygen Species, ROS）,因此ROS可能是各种胁迫信号的交叉点。近年来,科学家们通过DNA微阵列技术分析了植物体内大量受ROS调控的基因,对这些基因的功能进行了研究,并得到了许多在ROS信号转导中起重要作用的基因元件。实验室前期的研究发现,拟南芥GPX家族中的GPX3（Glutathione peroxidase 3）编码一个双功能蛋白,不仅能够清除植株体内过量的H₂O₂,而且可以直接或间接调节上游氧化信号,传递和激活下游信号分子;AtDOS1（Drought overly sensitive 1）则是通过酵母双杂交实验证实的能与GPX3发生强烈体外互作的蛋白因子,DOS1定位于细胞核中,具有核定位信号（NLS）和保守序列WWE domain（Trp-Trp-Glu）,在植物应答干旱和氧化胁迫中发挥重要作用。进一步的研究表明,DOS1可能为植物氧化胁迫信号转导途径中一个重要的转录调节因子,并且在H₂O₂胁迫下,DOS1会从细胞核向细胞质跨膜扩散,从而参与胁迫信号的传递。AtRRTF1（ROS related transcription factor 1）则是另一个通过芯片分析得到的可能与AtDOS1存在相互作用的基因,该基因编码带有锌指结构的转录因子,其表达受H₂O₂诱导,在生物及非生物胁迫信号转导中起中心作用。酵母双杂交实验也显示RRTF1和DOS1之间存在体外相互作用。但目前我们仍然缺少GPX3与DOS1以及RRTF1与DOS1之间存在体内互作的证据,并且对于DOS1在氧化胁迫下的跨膜运动及机理也不甚明了。本论文通过双分子荧光互补实验（BiFC）研究了GPX3与DOS1之间以及RRTF1与DOS1之间的体内互作关系,发现在野生型拟南芥叶肉细胞原生质体内,均能检测到这两对蛋白之间发生相互作用的黄色荧光信号,说明GPX3与DOS1以及RRTF1与DOS1都存在体内互作。进一步的DAPI染色实验证明DOS1与RRTF1发生互作的部位在细胞核内。在gpx3突变体原生质体中,没有检测到较为明显的RRTF1与DOS1之间互作的荧光,表明GPX3可能影响DOS1与RRTF1之间的相互作用。为确定GPX3与DOS1以及RRTF1与DOS1之间的体内互作是否影响氧化胁迫下DOS1的跨膜运动,从而影响DOS1传递胁迫信号,我们将实验室已有的DOS1::GFP转化WT植株（WT-DOS1::GFP）分别和gpx3,rrtf1 T-DNA插入纯合突变体进行杂交,筛选出带有DOS1-GFP标签的gpx3和rrtf1突变体植株（gpx3-DOS1::GFP和rrtf1-DOS1::GFP）,用激光共聚焦扫描电子显微镜分别观察氧化胁迫下两种突变体中DOS1的跨膜运动与WT中的差异,经过统计分析发现GPX3对DOS1的抗氧化胁迫运动有一定影响,在缺失GPX3的情况下,更有利于拟南芥下表皮条保卫细胞中的DOS1在受到H₂O₂和ABA作用时发生从细胞核向细胞质扩散的现象;而RRTF1表达下调时,DOS1的跨膜运动与正常情况相比没有明显的差别,说明RRTF1可能与DOS1的扩散关系不大。综上所述,GPX3与DOS1以及RRTF1与DOS1之间均存在体内互作,并且GPX3在一定程度上影响DOS1与RRTF1的体内相互作用;同时,GPX3与DOS1之间的体内互作可能影响DOS1在氧化胁迫下的运动。更多还原

【Abstract】 Adverse stresses can be classified as abiotic ones and biotic ones, which seriously affect plant growth and development. The abiotic stresses are diverse. They can trigger the production of reactive oxygen species （ROS）, which may be the cross point of various stress signals. In recent years, scientists have analyzed the expression patterns of large numbers of genes by DNA microarray, investigated the functions of the genes and found many important cis-elements acting in ROS signal transduction.Previous results from our laboratory revealed that GPX3 （Glutathione peroxidase 3）, a member of GPX family, encodes a functional protein. It can not only scavenge H₂O₂, but also regulate upstream ROS signals and activate and transfer downstream signals. AtDOS1 （Drought overly sensitive 1） has been identified to interact with GPX3 in vitro very strongly through the yeast two hybrid experiment. It localizes in nucleus, containing NLS and WWE domain, and plays a key part in plants response to drought and oxidative stress. DOS1 appears to be one of the important transcriptional regulatory factors in oxidative signal transduction in plants. Moreover, DOS1 can diffuse from the nucleus to the cytoplasm under H₂O₂ stress and transfer the redox signals. AtRRTF1 （ROS related transcription factor 1） is another gene which may interact with AtDOS1 in gene chips. It encodes a transcriptional factor containing zinc structure. The expression of AtRRTF1 is induced by H₂O₂ treatments. RRTF1 plays central roles in signal transductions of biotic and abiotic stresses. Yeast two hybrid experiment also showed that RRTF1 interacting with DOS1 in vitro. Yet, until now we still have no evidence about the in vivo interaction between GPX3 and DOS1 as well as RRTF1 and DOS1. We still have little knowledge about the diffusion mechanisms of DOS1 in Arabidopsis under oxidative stress.In this paper, the interactions between GPX3 and DOS1 as well as RRTF1 and DOS1 in vivo were investigated using the method of splite YFP （BiFC）. There was yellow fluorescence （interaction indicator） in protoplasts of WT plants transformed with GPX3-contained vector and DOS1-contained vector, as well as RRTF1-contained vector and DOS1-contained vector, indicating that DOS1 may interact with GPX3 and RRTF1 in vivo. DAPI staining analysis showed that the place in which DOS1 interacts with RRTF1 is the cell nucleus. No obvious interaction between DOS1 and RRTF1 in vivo was found in protoplasts from gpx3 plants suggesting that GPX3 may influence such interaction in vivo. To further determine if the interaction between DOS1 with GPX3 and RRTF1 affect the diffusion of DOS1 across nucleus membrane and the transferring of stress signals by DOS1 under oxidative stress, we obtained Arabidopsis gpx3 and rrtf1 T-DNA insertion mutants with DOS1-GFP tag （gpx3-DOS1::GFP, rrtf1-DOS1::GFP） by using hybridization technology between WT-DOS1::GFP and gpx3, rrtf1 mutants. Then, differences in DOS1 diffusion across nucleus membrane in the two mutants and WT were analyzed in confocal microscopy and statistical analysis. The results showed that GPX3 may influence the DOS1’s diffusing movement to a certain extent. Without the GPX3, the DOS1 in guard cells would be more inclined to diffuse from the nucleus to the cytoplasm after treatments with H₂O₂ or ABA. But when the expression of RRTF1 was down-regulated, there was no obvious difference for the DOS1 diffusing movement in rrtf1 mutant with DOS1-GFP tag compared to the WT with DOS1-GFP tag. These datas indicated that RRTF1 may not affect the diffusion of DOS1 under oxidative stress.To sum up, there exists in vivo interaction between GPX3 and DOS1 as well as RRTF1 and DOS1. GPX3 may affect the interaction between RRTF1 and DOS1. The in vivo interaction between GPX3 and DOS1 may modulate the diffusing movement of DOS1 across nucleus membrane under oxidative stress in Arabidopsis.更多还原