【作者】 宋兴舜；

【导师】 喻景权；

【作者基本信息】 浙江大学 ， 蔬菜学， 2006， 博士

【摘要】 设施园艺业已成为我国现代农业中的重要组成部分。其中,作为主体的设施蔬菜发展极为迅速。然而,全球性气候变化所引起的环境恶化、季节性或突发性的自然灾害以及病原菌的侵染等不利条件,给设施蔬菜的生产带来严峻挑战。这些逆境导致蔬菜品质下降,产量降低,直接或间接地造成巨大的经济损失。因此,深入探明蔬菜作物对逆境胁迫的响应机制,提高抗逆能力,培育具有良好抗逆特性的蔬菜品种,一直是各国蔬菜科研工作者关注的研究焦点。本文以黄瓜（Cucumis sativas L.）为试材,研究和探讨了非生物胁迫（高温、MV、H₂O₂或亚高温）对APX和SOD同工酶、组织水平或亚细胞水平下抗氧化系统的影响;研究和探讨了生物胁迫（CMV）对光合作用、呼吸作用及相应电子传递和亚细胞水平活性氧代谢的影响。主要研究结果如下: 1.在亚高温胁迫对不同基因型黄瓜SOD同工酶及抗氧化系统影响的研究中发现,不同基因型黄瓜对亚高温胁迫呈不同的抗氧化响应。耐高温品种叶片的光合机构,质膜等具有较高的耐高温能力,亚高温下生物量未受影响表明其在胁迫条件下仍具有较强的碳同化能力,电子流更多的用于碳同化,而用于产生ROS的电子流较少。ROS（如H₂O₂）相对含量较低,进而表现出多数抗氧化酶活性的钝化。但高温敏感黄瓜品种则相反,产生了大量的O₂^-,诱导其Cu/Zn-SOD和Fe-SOD的活性的增加,而并未引起Mn-SOD的变化。并在整体上提高了抗氧化系统酶活性及AsA,GSH的氧化还原状态。同时也上调了cAPX和CAT的转录水平,结合相应酶活性的变化,表明它们是在转录水平上的调控。这些变化对有效清除ROS均起到一定的积极作用。 2.研究了非生物胁迫（高温、MV及H₂O₂）对黄瓜APX同工酶和抗坏血酸（AsA）再生系统的影响。结果表明,在高温、MV及H₂O₂这三种胁迫的整个处理阶段,胞液APX（cAPX）、叶绿体基质APX（sAPX）和微体APX（mAPX）的活性变化呈现早期短暂降低,而后逐渐升高的趋势。与sAPX在活性水平上的变化高度一致,sAPX在转录水平也是呈先下调后显著上调的趋势,表明其也是在转录水平上的调控。与之相对,叶绿体类囊体膜APX（tAPX）的活性在整个实验阶段与对照相比均无显著差异,表现出一种组成性的表达变化。在AsA再生体系（AsA-GSH循环）中,单脱氢抗坏血酸还原酶（MDAR）、谷胱甘肽还原酶（GR）和AsA的氧化还原状态更多还原

【Abstract】 There is an increasing demand for vegetables with both high productivity and a superior quality in a rapid developing economy characterized with improved standards of living as in China currently. However, sustainance of high productivity and superior quality vegetables is imposing a big challenge on sound environmental management. Improper practices may lead to environmental deterioration which might induce global climate changes in the seasonal or paroxysmal natural disaster, and pathogen infection, etc. Therefore there is a practical need for individual or global researcher to elucidate clearly the response of vegetables to environmental stresses, attempts to improve their resistance, and to develop stress-tolerant cultivars through a combination of knowledge on vegetable physiology and breeding. In this study, we investigated the effect of abiotic stresses （such as heat, moderate heat, MV or H₂O₂） on APX isoenzymes, SOD isoenzymes, antioxidative systems in cellular or cub-cellular level in cucumber seedlings. Also, experiments were carried out to examine the relationship between photosynthetic, respiratory electron transport and antioxidant system in cell organelles in cucumber seedlings under a long-term cucumber mosaic virus infection （CMV）. The main results are as follows:1. The response of moderate-heat stress on SOD isoenzymes and antioxidative system was investigated in two （one tolerant and one sensitive） cucumber genotypes. Differences in changes between these two genotypes of cucumber seedling subjected to moderate-heat were observed. Moderate heat resulted in more intensive changes in antioxidative systems for the sensitive genotype. For sensitive genotype, moderate heat induced the activities of Cu/Zn-SOD and Fe-SOD. But no significant changes were observed in Mn-SOD activity. At the same time, moderate-heat resulted in a general increase of AsA-GSH cycle enzymes. Similarly, Consistent closely with their activities, the expression of cAPX and CAT were up-regulated, indicating that sAPX was regulated at the transcriptional level. These changes play a positive role in effectively weeding out redundant ROS.2. The effects of abiotic stresses （heat, MV and H₂O₂） on the APX isoenzymes and AsA regeneration system （AsA-GSH recyle） were examined in cucumber seedlings. The activities of cytosol APX （cAPX）, chloroplastic stroma APX （sAPX）, microbody APX（mAPX） increased after a slight decline throughout the experiment. Consistent closely with sAPX activity, the expression of sAPX followed a similar change pattern, indicating that sAPX was regulated at the transcriptional level. In contrast, constitutive expression was observed in thylakoid membrane APX （tAPX） activity and no significant changes in tAPX activity were found throughout the experiment. The increases in monodehydroascorbate reductase （MDAR） and glutathione reductase （GR） were accompanied with enhanced level of AsA/DHA, implying that the AsA regeneration system plays an essential role in compensating AsA degradation.3. The effects of methyl viologen （MV）-induced photo-oxidation on the activities of superoxide dismutase （SOD） and enzymes of the ascorbate-glutathione （AsA-GSH） cycle in chloroplasts, mitochondria and cytosol of cucumber leaves were investigated. Photo-oxidation by MV resulted in significant reductions in net photosynthetic rate （Pn） and increases in the ratio of the quantum efficiency of photosystem II （PSII）, Opsn to that of the quantum efficiency of CO2 fixation （Oco2）, followed by increased activities of SOD, and a general increase of AsA-GSH cycle enzymes in chloroplasts, mitochondria and cytosol. These increases were however, most significant in chloroplasts. There were also significant increases in dehydroascorbate （DHA）, reduced glutathione （GSH）, and oxidized glutathione （GSSG） except that the content of ascorbate （AsA） in chloroplasts and cytosol was slightly decreased and little effected, respectively. However, GSSG in mitochondria and GSH in cytosol were little influenced by the MV treatment. The activity of ascorbate oxidase （AO） in these organelles was independent of the MV treatment while the activity of L-galactono-l,4-lactone dehydrogenase （GLDH） in mitochondria was slightly inhibited by MV treatment. These results indicate that disturbance of electron transport in chloroplasts by MV influenced the metabolism of whole cell by a crosstalk signaling system and that the AsA-GSH cycle played a primary role in sustaining the levels of AsA.4. The effects of a long-term CMV infection on photosynthetic, respiratory electron transport and its relative response to ROS, together with the SOD and ascorbate-glutathione （AsA-GSH） cycle system were also investigated. Under a long-term CMV infection, reduced net photosynthetic rate （Pn） was accompanied by a lower quantum efficiency of PSII electron transport （<DPsn） mainly due to decreases in the photochemical quenching （qp） and the efficiency of excitation capture by open PSIIcenters （Fv’AFm’）. Further, CMV infection induced an increase in alternative electron flux （Ja）, probably leading to higher electron flux to O2. In mitochondria, Complex I and Complex II electron transports were inhibited whilst the AOX pathway was activated. In addition, the increases in H2O2 contents in chloroplasts and mitochondria were observed, followed by a general increase of SOD and AsA-GSH cycle enzymes in chloroplasts, mitochondria and cytosol. Taken together, it was concluded that the disturbance of photosynthetic and respiratory electron transports contributing, at least partly to the ROS such H2O2, influenced the metabolism of anti-oxidative systems in various organelles.更多还原