【作者】 王晓艳；

【导师】 陈伟；

【作者基本信息】 福建农林大学 ， 生物化学与分子生物学， 2013， 硕士

【摘要】 龙眼是我国南方名特优水果之一，主产于福建、广东、广西、台湾等省。龙眼果实由果皮、果肉(假种皮)、种子三部分组成，果肉肉质爽脆，清甜，营养丰富，具有较高的营养价值，可作为保健食品，被人们推崇为果中圣品，深受国内外消费者青睐。作为经济作物，我国龙眼的种植面积和产量分别占世界的70%和50%以上，是最大的龙眼生产国。然而，由于我国龙眼成熟期在8-9月高温季节，采后生理代谢旺盛，鲜果易变色、变质，其果实的贮藏和运输十分困难。果肉自溶是龙眼采后败坏的主要原因之一，严重影响其食用品质和商品价值，是限制龙眼长期贮藏和远距离运销的主要因素之一。而我国龙眼保鲜贮运技术的研究滞后，鲜果销售受到限制，难以大批量远销和出口，因此龙眼采后果肉自溶分子生理机制的研究在理论和生产上都有重要的意义。本研究运用蛋白质组学的方法，以“东壁”和“福眼”两个龙眼品种为试验材料，研究了两品种常温下分别贮藏0,1,2,3天后蛋白质的表达变化，使用软件ImageMaster分析共发现48个差异蛋白点的丰度变化达2倍以上，共成功鉴定了43个与龙眼果肉自溶相关的蛋白。随着贮藏天数的增加，“东壁”与“福眼”两品种分别有23与21个蛋白差异点，其中12个差异点属于两个品种共有；另外，8个蛋白只存在品种间差异，11个蛋白既存在品种间差异，也有同一品种不同天数的差异。这些蛋白分别参与了应激反应与解毒（40%）、能量与碳代谢（23%）、翻译调节（8%）、初级代谢与次生代谢（6%）、蛋白运输与降解（2%）、细胞骨架（2%）等。龙眼果肉自溶启动期与应激反应与解毒相关的蛋白质数量最多，这些蛋白质与抗氧化作用密切相关，所以抗氧化系统可能在龙眼果肉自溶发生期间发挥着关键性作用。除了谷氧还蛋白这类蛋白质全部下调，这说明龙眼果肉中接近衰老的组织，应对氧化胁迫的能力有所降低，抗氧化系统的抗氧化能力有所下降，保护细胞免受氧化性损伤的能力降低，不能有效的缓冲ROS积累引起的毒性作用，从而加剧果肉自溶的程度；在贮藏的整个过程中，“福眼”品种的蛋白质比“东壁”下调趋势更明显。能量和物质代谢是生物体的基本生命活动现象。一些研究认为果实采后衰老的发生可能与能量供应不足和生成速率下降有密切联系。本研究鉴定的与能量代谢相关的蛋白质主要参与糖酵解三羧酸循环、磷酸戊糖等途径，这些酶全部下调，这表明龙眼果实采后果肉衰老与自溶可能与能量供应不足有关，受ATP水平的影响。龙眼果肉自溶过程中，不仅能量代谢相关酶的表达量降低，且参与初级代谢途径（如核苷酸、氨基酸、氮的新陈代谢）的蛋白质、次级代谢物合成的蛋白质表达量也在减少。目前，关于龙眼果肉自溶的研究，主要还集中在形态和生理生化方面，对于其分子方面的研究还未发现报道。本研究从蛋白质组水平上研究与龙眼果肉自溶发生相关的蛋白质的表达模块及其功能，并对差异蛋白表达模块进行分类，阐明这些差异蛋白的生理功能及其表达变化与果肉自溶发生之间的相互关系。研究结果将为龙眼采后延缓果肉自溶的生产措施提供科学依据，并为龙眼抗果肉自溶品种育种奠定下分子生物学研究基础。更多还原

【Abstract】 Longan (Dimocarpus longan Lour.)is a typical subtropical non-climacteric fruit treebelonging to the family Sapindaceae that originated from South China. Longan fruit consists of athin, leathery and indehiscent pericarp surrounding a succulent edible aril with a large dark brownseed. Flesh aril is rich in carbohydrates, protein, fiber, fat, vitamin C, amino acids, and mineralswhich can be used as a possible supplement in the food. In recent years, the fruit aril which is alsoused in a wide range of medical applications, has exhibited excellent antioxidant ability and goodantityrosinase and anticancer activities. As an economic crop, China is the largest producer oflongan, the planting area accounted for70%of the world and the yield accounted for more than50%. However, storage and transportation of longan fruit is rather difficult. The fruit deterioratesrapidly after harvest, it have a very short shelf life of3-4days at ambient temperature due topericarp browning and aril breakdown irrespective of the cultivar. Post-harvest aril autolysis inlongan is a characteristic biological phenomenon in fruit deterioration and is a major factor thatdetermines fruit quality and marketing. While the technology of longan fresh-keeping research inChina lags behind, that lead to fresh fruit sales limited, mass products sales and exports over along distance are difficult. Therefore the molecular physiological mechanism of the aril autolysisresearch will be an important significance in theory and production.This study applied proteomics to research the protein expression changes of “Dongbi” and“Fuyan” aril after maked a storage of0,1,2and3days respectively under normal temperature.Then we use ImageMaster software analysis described above, and found48different proteinpoints whose abundance changes more than2times. At last43proteins were successfullyidentified associated with longan aril autolysis by making use of mass spectrometry techniques.There are23difference proteins in “Dongbi” and21in “Fuyan” with the increasing of storagedays,12different proteins be existed in two varieties; Additionally, eight proteins only existdifference between varieties;11proteins exist difference both between varieties and the samevariety in different storage days. Based on their physiological functions, all identified proteinswere grouped into the following nine groups: Stress response and defense; Energy and carbonmetabolism; Protein synthesis; Transport; Proteolysis; Biosynthesis of secondary metabolites;Cytoskeleton; Nucleotide metabolism; predicted protein. The largest functional class was associated with two categories: Stress response and defense(44%)and Energy and carbonmetabolism (26%), so the antioxidant system may play a key role during the mature fruit softening.These proteins all down-expression in addition to glutaredox what can explain that the ability tocope with stimulation from the outside and the antioxidant capacity of antioxidant systems intissue near aging is reduced. These results lead to the ability to protect cells from oxidativedamage is reduced so that longan aril autolysis increased. The down trend of proteins in “Fuyan”were more obviously than in “Dongbi” among the entire storage processes.This protein group devoted to energy and carbon metabolism is dominated by enzymes oftricarboxylic acid cycle, glycolysis and pentose phosphate pathway, which are expected to providethe necessary energy metabolites for the ongoing fruit ripening process, whereas were alldown-regulated, suggesting a decrease in carbon flow through these pathway during longan arilautolysis.In agreement with the decreased energy demand during aril autolysis process, not only Calvincycle, OPPP, glycolytic and other enzymes relative to energy metabolic levels were reduced, butalso protein species involved in primary metabolisms and pathways, such as metabolisms ofnucleotide, amino acid and nitrogen, and biosynthesis of secondary metabolites were decreased.At present, the research on longan aril autolysis mainly concentrated in aspects ofmorphology or physiological and biochemical, and the aspects of molecular has not been reported.The main purpose of this work is to study proteins expression modules and functions related in arilautolysis of longan at the level of proteomics, and we classify the protein expression module toexpound the relationship between the aril autolysis and the physiological functions and expressionchanges of these differential proteins. The research results will provide the scientific basis fordelay the aril autolysis of postharvest longan, and lay the molecular research foundation forbreeding antiautolysis longan varieties.更多还原