【作者】 云泽；

【导师】 程运江； 邓秀新；

【作者基本信息】 华中农业大学 ， 果树学， 2012， 博士

【摘要】 柑橘果实从采收到消费通常要经历一个漫长的过程,主要包括采后处理、贮藏、运输和销售等环节。在此过程中,由于生理失调、病原性病害以及机械损伤等因素引起的腐烂或变质往往造成巨大的经济损失。为了提高果实对贮运环境的抵抗能力、延长果实贮藏期限,同时兼顾对食品安全和生产环境的安全考虑,采后物理处理越来越引起人们的广泛关注。物理处理是柑橘采后品质保持的重要技术措施,目前柑橘采后物理处理的方法主要包括套袋、热处理、发汗处理、通风库贮藏、地窖贮藏和冷库贮藏等。虽然这些处理可以有效的延缓果实衰老,增加果实抗逆性,但是人们对物理处理改善柑橘果实采后贮藏性能的机理了解甚少。随着分子生物学和生物技术的快速发展,高通量的分析技术有可能为我们认识柑橘果实采后物理处理作用效果的调控机制提供重要的理论基础。为此,我们以椪柑(C. reticulata cv. EganNo.1)、HB柚(Citrus grandis×C. paradis)和温州蜜柑(Citrus unshiu Marc. GuoqingNo.1)为实验材料分别进行了低温贮藏、热处理和发汗等物理处理后,开展蛋白质组学、转录组学及代谢组学分析。主要结果如下：1、利用基于双向电泳和基质辅助激光解吸电离飞行时间质谱的比较蛋白质组学分析了椪柑在低温贮藏条件下不同贮藏时期的蛋白质表达变化。结果显示,共74个蛋白点在不同贮藏时期差异积累,其中56个蛋白点与绿色植物蛋白库中蛋白质相匹配。根据已有的分类信息将鉴定的蛋白质主要分为碳水化合物代谢、氨基酸代谢和响应环境逆境等。另外,我们对蛋白质的亚细胞定位进行了归类,并且对差异蛋白质在贮藏过程中的变化趋势进行了分类,发现果糖激酶、柠檬酸合成酶、顺乌头酸酶和苹果酸脱氢酶等蛋白质在调控果实贮藏过程中品质变化具有非常重要的作用。2、HB柚在不同贮藏温度中品质变化明显,我们分别利用数字表达谱和双向电泳对HB柚在不同贮藏温度下差异表达的基因和蛋白质进行了检测。结果显示,低温贮藏上调了果实中相应逆境基因的表达量,限制了果实内部信号转导、初生代谢和次生代谢速率、转运蛋白质活性。CBL-CIPK复合体可能参与了低温信号由果实外部向内部的传递；另外,柠檬苦素、诺米林素、甲醇和乙醛等物质在果实内部的积累,以及热激蛋白、14-3-3蛋白、COR15和冷胁迫响应基因和蛋白质的上调表达,从转录组学和蛋白质组学的角度为进一步解析果实响应低温逆境提供一个综合的视野。值得注意的是,低温贮藏果实中葡萄糖、果糖和脱落酸的含量显著降低(与常温贮藏相比),且大量生长素相关基因下调表达。由此可见,糖、生长素和脱落酸可能共同调控了果实的衰老。3、利用气相色谱-质谱联用仪和高效液相色谱-四级杆-飞行时间串联质谱仪对温州蜜柑采后热处理的果皮进行了初生代谢物和次生代谢物的检测,为进一步解析热处理诱导果实抗性提供了新的线索。结果显示,活性氧在热处理诱导果实抗性中起关键作用。热处理果皮中过氧化氢含量显著低于对照,直接增加了果实对逆境的忍耐度；黄酮类物质的增加直接参与了果实逆境的响应；消耗的过氧化氢用来合成了木质素,木质素的增加增厚了细胞壁,进而隔离了外部病原菌,减少了果实病害的发生。4、利用气相色谱-质谱联用仪和高效液相色谱-四级杆-飞行时间串联质谱仪对温州蜜柑发汗处理后果皮中初生代谢物和次生代谢物进行了检测,为解析发汗处理诱导果实抗性机理提供了代谢组学方面的信息。结果表明,水杨酸可能参与了发汗处理诱导抗性的信号传导,多数代谢物在发汗处理果皮中的积累与水杨酸处理果皮中的积累趋势完全一致,如糖类物质(阿拉伯糖含量下降,其余均增加)、有机酸类物质(羟基丙酮酸含量下降,其余均增加)、脂肪酸类物质(含量均增加)、糖醇类物质(含量均增加)和其它酸类物质(含量均增加)更多还原

【Abstract】 Citrus fruits, from harvest to human consumption, it require a length storage period for postharvest treating, storing, shipping and marketing. During that time, the main damages of fruit are coming from physiological disorders, pathogenic disease and mechanical damage. As more attention was paid in food safety and environmental pollution, postharvest physical treatment become into the first choice instead of chemical treatments. Postharvest physical treatments are used to inhibit fruit pathogens, reduce fruit weight loss and increase fruit resistibility during postharvest storage, containing bagging, sweating, heat treatment, cold storage, gas controlling storage, and so on. However, less information about the mechanism is available. Along with the technological progress of proteomics, transcriptomics and metabolomics, high-throughput analysis technology provide important theoretical basis for understanding the regulatory mechanism of postharvest physical treatment induced citrus fruit resistance. In present study, ponkan (C. reticulata cv. Egan No.1) fruits, Hirado Buntan pummelo (HBP; Citrus grandis x C. paradis) fruits and’Kamei’Satsuma mandarin(Citrus unshiu Marc. Guoqing No.1) fruits were chosen to explore the mechanisms of postharvest treatments induced fruit resistibility and delayed fruit senescence during postharvest storage. Main results were following.1. Comparative proteomics analysis was carried out in’Egan No.1’ponkan fruit at differential storage periods. Two-dimensional gel electrophoresis (2-DE) coupled Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) were performed to examine the protein changes during the postharvest storage period. Results showed that74proteins were differentially regulated, from which56proteins were identified by blasting against NCBInr (green plant) and EST viridiplantae databases. All identified proteins were then classified into functional classes according to known biosynthetic pathways, including C-compound and carbohydrate metabolism, amino acid metabolism and response to storage environmental stimuli. In addition, subcellular location and time-dependent accumulation trends of differentially accumulated proteins associated with fruit quality were analyzed. The result showed Fructose kinase, citrate synthase, aconitase and malic dehydrogenase play key role in fruit quality regulation during storage.2. HBP fruit were chosen to explore the mechanisms that maintain citrus fruit quality during lengthy LT storage using transcriptome and proteome studies based on digital gene expression (DGE) profiling and2-DE, respectively. This study showed that LT up-regulated stress-responsive genes and arrested signal transduction, primary metabolism, secondary metabolism and the transportation of metabolites. Calcineurin B-like protein (CBL)-CBL-interacting protein kinase (CBL-CIPK) complexes might be involved in the signal transduction of LT stress. In addition, the accumulation of limonin, nomilin, methanol and aldehyde, as well as the up-regulated heat shock proteins,14-3-3protein, COR15and cold response-related genes provided a comprehensive proteomics and transcriptomics view on the coordination of fruit low temperature stress responses. Fruit quality is likely to be regulated by sugar-mediated auxin and abscisic acid signaling, such as down-regulation of carbohydrate metabolism, nitrogen metabolism, lipid metabolism and secondary metabolism.3.’Kamei’Satsuma mandarin fruits were chosen to explore the mechanisms of HT induced fruit resistibility during postharvest storage using metabolome studies based on Gas chromatography coupled to mass spectrometry (GC-MS), High performance liquid chromatography hybrid quadrupole time-of-flight mass spectrometry (HPLC-QTOF-MS). This study has provided new insights into HT induced fruit resistance. Interpretation of the data for metabolites revealed that reactive oxygen species (ROS) was paly a vital role in HT fruit stress resistance. The intracellular H2O2content was decreased in HT fruits, which might increase fruit resistibility in response to external stress. In addition, Flavonoids were up-regulated in HT fruits, which directly involved in the response to external stress. Moreover, the decreased H2O2was used for synthesis of lignin, which lead to the content of lignin was increased in HT fruits. The increased lignin thickened cell wall, which was involved in isolated external pathogens from fruits.4.’Kamei’Satsuma mandarin fruits were chosen to explore the mechanisms of ST induced fruit resistibility during postharvest storage using metabolome studies based on GC-MS and LC/QTOF-MS. This study has provided new insights into ST induced fruit resistance. Interpretation of the data for metabolites revealed that salicylic acid (SA) was paly a vital role in ST fruit stress resistance. Many metabolites were up-accumulated in ST pericarp, such as sugars (except arabinose), organic acids (except Hydroxypyruvic acid), fatty acids, sugar alcohols, and other acids. All those changes in metabolites were correlated well in SA treated pericarp.更多还原