【作者】 王国泽；

【导师】 茅林春；

【作者基本信息】 浙江大学 ， 食品科学， 2006， 博士

【摘要】 磷脂酶D(PLD)是植物中一类主要的磷脂水解酶。它的作用不仅在于通过水解细胞膜中的磷脂影响膜的结构、功能和稳定性,而且在细胞信号转导,脂质代谢,膜运输以及生物膜形成等方面发挥着重要作用。近年来,有关PLD在跨膜信号转导中的作用成为新的研究热点。本研究的目的是为了探明PLD在黄瓜冷害发生与发展中的作用,调查热处理减轻黄瓜贮藏冷害的原因是否与PLD活性变化、PLDmRNA的表达规律有关。 热处理是一种简单易行的物理处理方法,由于它具有无毒无害、无环境污染、无化学药物残留等优点,因此具有十分广阔的应用前景。前人研究证明热处理可诱导合成小分子量的热激蛋白,减轻和抑制冷敏型果蔬冷害的发生,提高产品的贮藏性能。本研究采用热处理的目的是反向验证PLD在黄瓜冷害发生与发展中的作用。 黄瓜(Cucumis sativus L.)在低于10℃的温度下贮藏容易产生冷害。本研究以冷敏型植物材料一黄瓜(津优1号)为试材,采后将黄瓜分为两组:一组(冷胁迫组)置于20℃、90%RH人工气候箱24 h,另外一组(热处理组)置于37℃、90%RH人工气候箱中预热24h,然后贮藏于2℃。为了研究PLD在黄瓜冷害发生和发展及热处理减轻黄瓜冷害中的作用,分别在贮藏第0,3,6,9,12,15天对贮藏过程中各种生理生化指标变化进行分析测定:同时,在热处理组黄瓜开始出现冷害症状时(本研究为贮藏第9天)将黄瓜横切为上(靠近花萼端)、中、下(靠近茎端)三部分并对相关生理生化指标进行研究以分析PLD在转导黄瓜冷害信号中的作用,探讨冷胁迫条件下黄瓜PLD变化引起冷害发生的作用机理。 本研究通过克隆及鉴定获得了889个核甘酸对长黄瓜PLD的保守域片段。对获得的片段进行氨基酸序列及进化树分析,结果表明,黄瓜中的PLD应该归属PLDα,在比对的12种植物中,黄瓜与蓖麻同源性最高。 本研究表明低温胁迫条件下PLD和LOX的激活启动了黄瓜感应的机制,黄瓜果实冷害的初始反应可能是由于PLD和LOX的激活引起细胞膜的降解造成的。磷脂降解酶类的激活可能是冷害初始反应的首要步骤。黄瓜开始出现冷害症状时,上、中、下不同部位冷害指数、电导率、MDA含量、PLD活性、LOX活性、PA的含量以及PLD mRNA表达量之间都存在明显的差异。说明2℃贮藏期间不可逆转的冷害首先是从靠近花萼端的上部发生,然后经过中部向靠近茎端的下部逐渐发展。而PA含量的梯度差异可能是造成黄瓜不同部位冷害从上部到下部逐渐发展的直接原因。 2℃贮藏期间黄瓜果实中呼吸速率和乙烯释放量下降趋势具有良好的一致性,对冷藏过程中更多还原

【Abstract】 Phospholipases D (PLD EC 3. 1. 4. 4) is a key enzyme that catalyzes the hydrolysis of membrane phospholipids into phosphatidic acid (PA) and a hydrophilic headgroup, which are the backbones of biological membranes. The activities of these enzymes not only have an important impact on the structure and stability of cellular membranes but also play an pivotal role in regulating many critical cellular functions, including cellular signal transdcution, lipid metabolism, vesicle trafficking, cell proliferation, celluar backbone formation and sensescence. In recent years, the role of PLD in transmembrane signal transduction has become a new research topic. The objectives of this study were to evaluate the function of PLD in incidence and development of chilling injury (CI) and the effectiveness of warm treatment treatment to reduce CI and storage physiology, and whether these effects were related to changes in PLD activity and PLDmRNA expression.Warm treatment is an easy and feasible physical treating method. It has precious future for innocuity and non-pollution without deposit of any chemistry on fruits and vegetables. Several previous studies have suggested that warm treatment that increase chilling tolerance is thought to work through the induced synthesis and accumulation of specific heat-shock proteins (hsps), and also for improving storability. Application of warm treatment in this study is to test and verify the role of PLD in incidence and development of CI inversely.Cucumber fruit (Cucumis sativus L) is chilling sensitive and susceptible to CI when fruit are stored at temperatures of less than about 10℃. This paper selected one variety of cucumber "JINYOU-1" as testing material which was sensitive to chilling. Before 2℃ storage, cucumbers were devided into two groups, one was treated in 20℃, 90% RH for 24h (chilling stress group), the other was treated in 37°C, 90% RH for 24h (warm treatment group). In order to investigate the metabolic mechanism of CI and effect of warm treatment on inhibiting CI by regulating PLD in cucumber during low temperature storage, physiological and biochemical changing, PLDmRNA expression, changes of fatty acid in membrane were evaluated after storaged at 2°C for 0, 3, 6, 9, 12, 15 d, respectively. The article also gives a brief introduction on the role of PLD in cucumber cellular CI signal transduction. For analysis, when CI symptom first appeared in warm treatment group, sample fruits were cut into three sections with equal length, namely top (calyx end), middle and bottom (stalk end), respectively. Thus, we can . understand the mechanism of CI induced by the changes of PLD in chilling stress.In this study, a partial PLD gene, encoding a putative phospholipase D, was identified in cucumber fruit. This cloned PLD fragment had 889 bp nucleic acids and the deduced amino acid sequence showed high identity with known PLD genes. Phylogeny analysis of PLD amino acid sequences based on cDNA clones from various plant species revealed that identities in cucumber PLD shared the highest identity (81%) with that of caster bean and belonged to PLDa class.In general, chilling stimulated lipid peroxidation and PLD action in chilling stress group cucumber fruit prior to the development of irreversible injury. Both lipid peroxidation and PLD action should be responsible for creating stress conditions in cucumber fruits.Thus, activation of enzyme hydrolysis of membrane phospholipids may be primary step for initiation of CI. When CI symptom first appeared, there is significant difference between CI index, electrolyte leakage, MDA content, PLD and LOX activity, PA content and PLDmRNA expression in top, middle and bottom, indicating irreversible CI occurred initially in the top area near calyx of fruits, and developed toward the bottom near stalk. We suggest that PLD and LOX are associated with the initiation of CI by involving in membrane deterioration and signaling pathway in response to chilling stress, while the difference in PA content may be direct reason.In the study, the change of respiration rate shared similar trends with ethylene production during storage at 2*C. The correlation analysis indicated that excessive accumulation of PA is main reason forCI occurrence.The relationships between CI and physiologicaland biochemical factors are complicated. Changes of Ca2+ content especially membrane-associated Ca2+ content play a vital role inalleviation of CI by warm treatment treatment. The result showed that warm treatment decreased CI index, maintained low level of PLD and LOX activity, inhibitted PLDmRNA expression, resulted in lower cell membrane permeability and PA content, reduced MDA and proline accumulation and delayed the decrease of soluble protein contents. Above all, chilling stress decrease content of membrane-associated Ca2+, which increased PLD mRNA expression firstly, leading to high level of PLD activity;at same time, relative higher levels of cytosolic Ca2+ concentrations could increase to lethal levels, thus activating autocatalytic membrane lipid degradation and LOX activity. As a result, PLD, cooperating with LOX make PA content increased. That may be the main reason for initiation of CI.更多还原