【作者】 孟艳艳；

【导师】 喻树迅；

【作者基本信息】 中国农业科学院 ， 作物遗传育种， 2011， 博士

【摘要】 为了研究短季棉早衰的机理及一氧化氮(NO)对短季棉叶片蛋白质组的影响,本试验选用2个早熟不早衰(中棉所16和辽4086)和1个早熟早衰(中棉所10)的短季棉品种作为试验材料,对大田条件下叶片衰老不同阶段的各种生理指标、抗氧化物酶活性及编码基因和室内条件下叶片衰老中NO对抗氧化物酶的作用进行了研究;同时利用非定量标记法分析了NO对棉花叶片蛋白质组的作用和影响。主要研究结果如下:自然条件下,35天是早衰和不早衰两种短季棉材料叶片衰老发生差异的一个关键时期。早衰材料中H2O2和MDA的含量高于不早衰材料;NO含量在衰老叶片中迅速降低,同时早衰材料中NO含量更低且下降的更快,NO可能是延缓叶片衰老的一个重要因素。早衰类型材料中AsA的含量低于不早衰材料,GSH含量在叶片的整个生育进程和不同材料之间都没有显著的差异,这暗示着叶片衰老过程中AsA在清除ROS方面比GSH发挥着更为重要的作用。不早衰材料中CAT的活性显著高于早衰材料,基因的表达量变化和酶活性一致。APX活性差异出现在35至50天之间,基因的表达量和酶活性的趋势相似。Mn-SOD在短季棉叶片衰老过程中没有明显的变化,Fe-SOD基因的相对表达量在三个材料中表现趋势相同且没有显著差异。eCu/Zn-SOD和cCu/Zn-SOD在清除ROS和延缓衰老方面比ChlCu/Zn-SOD发挥着更为重要的作用。总的POD活性随着叶片的衰老而迅速增加,POD可能并不清除H2O2而是起到产生H2O2的作用。室内条件下,外施SNP溶液后的植株,其NO含量在子叶的整个生育期都比对照组高且差异显著。在同一时期,处理组中CAT和APX的活性显著高于对照组,在生育后期表现的更为明显。外施SNP可显著降低参试品种POD的活性和相关基因的表达。外源NO对SOD的活性有先抑制后促进的作用。不同类型的SOD对NO的反应不同,Cu/Zn SOD的反应最敏感,其中又以cCu/Zn SOD基因的作用更为突出。NO通过调控植株体内一系列的CAT、APX、POD和SOD等氧化/抗氧化系统,延缓叶片的衰老进程。本试验首次利用非定量标记法分析不同浓度的NO对棉花叶片蛋白质组的影响,一共得到了121个差异表达蛋白,这些蛋白按照功能分为13类,已知定位的蛋白分布在叶绿体、高尔基体、细胞质和线粒体中。信号通路分析发现,NO广泛地参与细胞的各种生理活动,并且对光合作用、氧化磷酸化和蛋白质加工过程产生了明显的调控作用。高浓度的NO会对细胞产生毒害作用,并且这种毒害作用可能是通过抑制光合元件的表达、减弱ATP合成能力及引起蛋白质错误折叠和装配而产生的。更多还原

【Abstract】 To study the mechanism of early senescence and the effect of NO on leaf proteome in short season cotton, 2 cultivars with non-premature senescence (CCRI16 and Liao4086) trait and 1 clutivar with premature senescence (CCRI10) trait were selected as the experiment samples. Under field condition, various physiological parameters,activity of anti-oxidative enzymes and their corresponding genes were investigated at different stage of leaf senescence; under indoor condition, the effect of NO on anti-oxidative enzymes were also studied. Meanwhile, the effect of NO on cotton leaf proteome was also analyzed by using label-free method. The results are as follows:Under natural condition, the 35 day can be regarded as a watershed for the onset of leaf senescence in the two genotypes. The content of H2O2 and MDA was higher in the premature senescence (PS) genotype than in the non-premature senescence (NPS) plants; the content of NO decreased sharply and was significantly lower in PS than that of NPS cultivar at the late stage of leaf senescence, hence NO might be important factor delaying leaf senescence.The AsA concentration was lower in the PS genotype when compared with NPS genotype, and no significant distinction of GSH in both genotypes was observed throughout the whole lifespan of leaf development and among different materials. It is suggested AsA plays a more crucial role than GSH in terms of ROS scavenging during cotton leaf senescence. The activity of CAT was significantly higher in the NPS plant than in the PS plant, and change in its relative mRNA expression of CAT was consistent with enzyme activity. The activity of APX differed significantly between the NPS and PS from 35- to 50-days and the relative mRNA expression level of APX exhibited the similar pattern with its coresponding enzyme activity. No significant change in the relative expression level of Mn-SOD during leaf aging was observed; throughout the whole leaf development, there was also no significant difference of the relative expression level of Fe-SOD among the three cotton genotypes. The cCu/Zn-SOD and eCu/Zn-SOD play more pronounced roles than chloroplastic Cu/Zn-SODs in terms of ROS scavenging and delaying senescence. Overall POD activity in leaf increased strikingly when undergoing natural senescence, therefore it is suggested that POD did not act to eliminate H2O2 but involved in H2O2 generation.Under indoor condition, after exogenous application of SNP, the NO content was significantly higher in the treatment group than that of control group during the whole cotyledon development stage. At the same stage, the activity of CAT and APX in treatment group was significantly higher than that of control group, especially at the late stage of cotyledons. The activity of POD and its gene expression substantially declined in the subject cultivar by exogenous spray of SNP. Although exogenous NO could inhibit the activity of SOD at early stage of cotyledon development, the treatment group demonstrated greater SOD activity than that of control group with the advancement of leaf senescence. The responses to NO varied among different types of SOD, and the Cu/Zn SOD was the most sensitive isoforms among which cCu/Zn SOD’s genes played a more potent role. The physiological and molecular mechanism underlying the delaying effect of NO on leaf senescence is thus revealed by fine coordination of the activity of oxidation and anti-oxidation systems (CAT, APX, POD and SOD) in plant.To date, it is the first time that the effect of different concentrations of NO on the leaf proteome in cotton was investigated using the label-free approach. In this study, 121 differentially expressed proteins were obtained which were functionally divided into 13 groups and spread accross chloroplast, Gogi apparatus, cytoplasm and mitochondria. The pathway analysis demonstrated that NO involved in various physiological activities and had a pronounced impact on photosynthesis, oxidative phosphorylation and protein processing. High concentrations of NO could constitute a toxic threat on cells and the toxic effect may be induced by inhibiting expression of photosynthetic components, decreasing the capability of ATP synthesis and leading to the misfold and assembly of proteins.更多还原