【作者】 张扬；

【导师】 周艳虹；

【作者基本信息】 浙江大学 ， 园艺学， 2013， 硕士

【摘要】 全球气候变暖导致设施蔬菜高温减产问题日益突出,研究设施蔬菜的耐高温机制对于改进栽培措施和耐高温品种选育具有重要意义。一氧化氮(NO)作为植物抗逆/抗病反应的第二信使,通过蛋白S-亚硝酰化修饰完成整个信号转导过程。S-亚硝酰化调控植物的高温抗性,但具体机制仍不清楚。本研究以野生型(Solanum lycopersicum Mill.cv.Ailsa Craig)为材料,利用VIGS技术专一性的沉默了SlGSNOR、SlRBOH基因,以沉默植株为材料,研究了高温下番茄SlGSNOR的沉默对植株光合机构和高温抗性的影响,并通过抗氧化系统、基因表达和激素含量的分析,研究了S-亚硝酰化在植物抗逆反应尤其是耐高温反应中的作用机制,为改进高温条件下番茄的栽培措施和耐高温新品种创制提供理论基础。主要结论如下：1研究了高温下番茄SlGSNOR基因沉默对光合参数、Rubisco活化酶以及荧光参数的影响。高温胁迫下,发现pTRV-SlGSNOR沉默植株与pTRV植株相比,高温下叶片净光合速率下降,净光合速率的下降伴随着Gs的下降及Ci的上升。沉默植株的Rubisco酶活力降低。与pTRV处理植株相比,沉默植株Fv/Fm比值、PSⅡ实际光量子产量(ΦPSⅡ)、光化学猝灭系数(qP)降低,NPQ受到抑制。2研究了高温下胁迫下GSNOR的响应及对NO和SNOs的影响。高温处理3h, GSNOR酶活力提升最明显,pTRV-SlGSNOR植株pTRV植株均有明显提升,而pTRV-SlGSNOR沉默植株的活力显著弱于pTRV。与对照(CK)相比,高温处理(HT)后,pTRV和pTRV-SlGSNOR植株的SNOs受到诱导；随着处理时间的增加,pTRV-SlGSNOR的提高幅度更大,至高温处理后24小时,pTRV-SlGSNOR沉默植株为pTRV的1.5倍。与此同时,pTRV和pTRV-SlGSNOR植株NO在高温处理后逐渐提升,沉默植株的NO提升幅度明显高于pTRV。高温后番茄SlGSNOR沉默导致内源的SNOs和NO不能及时被清除而逐渐累积。3研究了高温下番茄SlGSNOR基因沉默对ROS的影响。H2O2/NO作为第二信使参与激素应答和抗逆反应。高温下番茄SlGSNOR基因沉默使得H202诱导受到抑制,不能启动活性氧“预警机制”,抗氧化酶活性及基因表达降低,谷胱甘肽受到影响,MAPK级联反应受到抑制,进而导致MDA含量显著增加,Fv/Fm明显降低。这表明SlGSNOR对维持redox信号平衡和增强氧化胁迫抗性具有重要作用。研究了高温下番茄SIGSNOR基因沉默对激素的影响。SIGSNOR的缺失对耐热性的影响与ABA、SA有关。4研究了S-亚硝酰化在高温胁迫下ROS和NO信号交叉中的作用机理。番茄pTRV-SlGSNOR沉默植株遭受高温逆境时,H2O2诱导受到抑制,表现严重的氧化胁迫,外源喷施H2O2后抑制有所缓解。外源施加H2O2后,沉默植株的氧化胁迫程度有所缓解,高温抗性增强,膜脂过氧化程度减轻。并且,外源喷施H2O2后,氧化还原状态有所改善,进而导致抗氧化酶活力的提高及抗氧化基因表达水平的提高。pTRV-SlRBOH沉默植株在高温下不能诱导ROS的爆发,GSNOR酶活力没有被诱导,造成沉默植株SNOs不断累积,不能被清除,高温下体内的响应趋势类似于pTRV-SlGSNOR。NO可能通过S-亚硝酰化抑制NADPH氧化酶,从而阻断ROS-MAPK-激素-防御基因表达的信号通路。GSNOR介导的S-亚硝酰化是番茄耐高温反应中调控ROS和NO信号交叉应答关键机制。更多还原

【Abstract】 Yield reduction issues of greenhouse vegetable have become increasingly prominent due to global warming. The research of high temperature resistant mechanism for greenhouse vegetables is important for the improvement of cultivation practices and high temperature resistant breeding. Nitric oxide (NO) as a plant second messenger of disease-resistance/resistance response, complete the signal transduction by the S-protein nitrosylation. S-nitrosylation regulates the high temperature resistance of plants, but the specific mechanism is still not clear.In this study, TRV and tomato plants v(Solarium lycopersicum Mill.cv.Ailsa Craig) were used to construct the gene silencing vecor with the pragment of SIGSNOR, SIRBOH. The VIGS plants were used to investigate the effect of tomato SIGSNOR silencing under high temperature on the photo synthetic apparatus and high temperature resistance. Then we analysed the gene expression and hormone content, antioxidant system to study the the role of S-nitrosylation in plant resistant response particularly high temperature resistence, providing theoretical basis for improving cultivation practices under high temperature. The main conclusions are as follows:1The Effects of SIGSNOR gene silencing under high temperature on photo synthetic parameters, Rubisco activity and fluorescence parameters were investigated. Under high temperature stress, the pTRV-SlGSNOR silencing plants decreased photosynthetic rate compared with pTRV.The decrease of photosynthetic rate accompanied with the decrease of Gs and the increase of Ci. The Rubisco activity decreased in the pTRV-SIGSNOR silencing plants. In the gene silencing plants Fv/Fm、φPSⅡ、qP decreased significantly,and NPQ was inhibited.2We have studied GSNOR activity and the effect on the NO and SNOs under hith temperature stress. After high temperature treatment for3hours. GSNOR enzyme activity increase the most, both pTRV-SIGSNOR and pTRV have improved significantly, while the pTRV-SIGSNOR silencing plants showed significantly lower than pTRV.After heat sress, the SNOs of pTRV and pTRV-SIGSNOR plants were induced. As the increase of treatment time. pTRV-SlGSNOR was increased more than pTRV, and after high temperature treatment for24hours, the SNOs of silencing plants is1.5times of that of pTRV. At the same time, the NO contens of pTRV and pTRV-SlGSNOR increase gradually after the high temperature treatment, NO increase rate of silencing plants was significantly higher than that of pTRV. Therefore, because of SlGSNOR silencing, tomato endogenous SNOs and NO can not be timely cleared under high temperature.3The impacts of tomato SlGSNOR gene silencing on ROS under high temperature stress were examined. H2O2/NO is a second messenger involved in hormone response and stress response.After high temperature,tomato SlGSNOR gene silencing inhibited the induction of H2O2, so reactive oxygen species as a "early warning mechanism" can not start, resulted in an decrease of antioxidant enzyme activity、gene expression and glutathione content. The induction of MAPK cascade was inhibited.Thus led to a significant increase in the content of MDA. This indicates that the SlGSNOR play an important role in maintaining the balance of redox status and enhancing oxidative stress resistance.4The influences of SlGSNOR gene silencing on hormone under high temperature were studied. The effect of SlGSNOR gene silencing on heat resistance is related to ABA and SA.5The role of S-nitrosylation in the ROS and NO signals under high temperature. After high temperature treatment, in the pTRV-SlGSNOR silencing plants, H2O2induction was inhibited, which led to severe oxidative stress. Exogenously applied H2O2, the inhibition eased. The redox status improved, leading to increase the activity of antioxidase and antioxidant gene expression level. The pTRV-SlRBOH silencing plants could not induce the outbreak of ROS under high temperature, and the GSNOR enzyme activity was not induced, leading to SNOs accumulation. This showed a similar response as pTRV-SlGSNOR.Perhaps NO inhibited NADPH oxidase through S-nitrosylation, thereby blocking the ROS-MAPK-hormone-defense gene expression signal pathway.更多还原