【作者】 赵宇瑛；

【导师】 茅林春；

【作者基本信息】 浙江大学 ， 食品科学， 2011， 博士

【摘要】 新鲜果蔬在采收、采后处理以及加工过程中不可避免地会受到机械损伤,机械损伤对植物而言是最严重的胁迫之一,损伤信号能够从损伤组织传递到未损伤组织,从而产生一系列的生理学反应。近年来,对果蔬采后机械损伤的研究已经成为人们关注的焦点。采后黄瓜遭受机械损伤期间,可以诱导黄瓜产生“伤乙烯”,乙烯的过量生成可加速黄瓜组织生理代谢,从而引发黄瓜产生“伤呼吸”,进一步加速黄瓜组织的衰老。同时,机械损伤可诱导细胞膜上的钙离子发生变化,钙离子在摩尔级含量变化的情况下即可起到调节PLD酶活性的作用。损伤后随着细胞膜钙离子含量的减少,黄瓜PLD酶活性增强,从而促进了细胞膜磷脂的水解作用,细胞膜PC含量明显减少,PA含量显著增加,而在这一过程中PI含量基本稳定,表明PLD可将PC水解为PA。在损伤的过程中LOX活性增强,进一步催化了细胞膜脂质过氧化。在PLD与LOX两种酶的共同作用下,使细胞膜不断降解,引起黄瓜电导率在损伤后持续增大,表明黄瓜细胞膜的完整性已经遭受损害。因此,我们推测PLD以及LOX参与了采后黄瓜对机械损伤的响应,可引起组织内部的一系列生理变化。由于黄瓜各部位成熟度不同,机械损伤对果实不同部位造成的影响不尽相同。本文以黄瓜不同空间部位为研究对象,分析了黄瓜不同部位对机械损伤的响应。机械损伤可诱导黄瓜果脐、中部、果肩部位组织中“伤乙烯”和“伤呼吸”的产生,并且导致黄瓜“伤乙烯”释放量增大、“伤呼吸”增强、电导率增大,PLD、LOX活性提高,并且随PLD活性的增强,PC、PI含量降低,PA含量升高。机械损伤还可诱导细胞膜钙含量降低。研究结果同时表明,受损黄瓜果脐部的“伤乙烯”、“伤呼吸”、电导率、PLD活性、LOX活性明显高于其他部位,并具有比其他部位低的细胞膜钙含量。本文研究了机械损伤对黄瓜抗氧化系统的影响,结果表明,机械损伤能诱导黄瓜中O2·-和H2O2的增加；随着损伤时间的延长,损伤黄瓜O2·-和H2O2含量呈现先上升后下降再上升的趋势；损伤黄瓜SOD、POD、CAT、APX、MDHAR变化趋势均表现为先上升后下降的趋势；损伤黄瓜DHAR酶活性变化表现为升高,稳定并缓慢过渡到下降的过程。在损伤过程中,损伤黄瓜的各类抗氧化酶的活性均高于对照。黄瓜GR酶活性变化为先上升再下降最后上升的趋势,变化幅度比较平缓,且损伤期间GR酶活性始终低于对照。损伤期间,损伤黄瓜的ASA、GSH含量均低于对照,而DHA和GSSG含量均高于对照。机械损伤诱导黄瓜各部位产生超氧阴离子以及H202,其含量由大到小的顺序均为果脐部最大,中部次之,果肩部最低；各部位衰老程度的不同会引起抗氧化酶活性的变化存在差异,黄瓜果脐部SOD、POD、CAT、MDHAR、APX、DHAR活性分别大于中部和果肩部,黄瓜GR酶活性不同于其他酶活性,各部位活性顺序为：果脐部酶活性最小,中部次之,果肩部最高；ASA和GHS的变化由低到高的顺序为：果脐部<中部<果肩部,损伤黄瓜各部位的ASA和GHS含量均低于对照；黄瓜各部位DHA和GSSG含量与ASA的变化相反,高低顺序为：果脐部>中部>果肩部；损伤黄瓜果脐部的DHA与GSSG含量明显高于对照,导致各部位GSH/GSSG高低顺序为：果肩部>中部>果脐部,且损伤黄瓜各部位GSH/GSSG明显低于对照黄瓜。机械损伤可引起黄瓜PLD和LOX以及抗氧化物酶基因表达量上调,且具有时间效应。损伤黄瓜中PLD和LOX酶基因的表达量均明显高于对照。SOD、POD、CAT、APX、MDHAR、DHAR酶基因在损伤前期表达量较高,高于对照；到损伤后期(8-10 h)其表达量出现降低的现象,表达量低于对照。GR基因表达量的变化趋势与其他抗氧化酶基因表达趋势一致,但其表达量在损伤期间始终低于对照。损伤黄瓜各部位的PLD、LOX、SOD、POD、CAT、APX、MDHAR、DHAR基因表达量均高于对照,且由大到小的顺序均为果脐部最大,中部次之,果肩部最低；GR基因的表达量与其他酶基因的表达量变化趋势相反,由大到小的顺序均为果肩部最大,中部次之,果脐部最低,且均低于对照。实验证明,抗氧化酶基因表达量与黄瓜不同部位的成熟度相关。更多还原

【Abstract】 Mechanical damage is considered as a type of stress, which is also the main cause of losses in the postharvest horticultural products. It can produce several signals that migrate through injury tissure into uninjured tissue and induce some physiological responses. In rescent years, the research on responses of mechanical wounding in postharvest horticultural products is highly attentioned.This study was to investigate the response of phospholipase D (PLD), lipoxygenase (LOX) and antioxidant system to mechanical wounding in postharvest cucumber (Cucumis sativus L. cv. Biyu-2) fruits. "Wound respiration" and "wound-induced ethylene", membrane-associated Ca2+ content, activities of PLD and LOX, contents of phosphatidylcholine (PC), phosphatidylinositol (PI), phosphatidic acid (PA) and electrolyte leakage were determined in cucumber fruits following mechanical wounding. Results show that "wound respiration" and "wound-induced ethylene" were induced by mechanical wounding. PLD and LOX activities increased upon wounding, while membrane-associated Ca2+ content decreased. Accompanying with the increase of PLD and LOX activities, accumulation of PA and loss of PC and PI were observed in all fruits, at the same time, higher electrolyte leakage was observed with activities of PLD and LOX increased. Results suggest that PLD and LOX might be the main hydrolytic enzymes of phospholipids in postharvest cucumber fruits participating in the mechanical wounding injury.Individual cucumber fruit were wounded via puncture in the navel, middle and neck tissues. Changes of "wound respiration" and "wound-induced ethylene", lectrolyte leakage, activities of PLD, LOX, contents of PC, PI, PA and electrolyte leakage, membrane-associated Ca2+ content were found in navel, middle and neck tissues of cucumber fruits at 4 h after wounding. Both "wound respiration" and "wound-induced ethylene" was significantly higher in navel end than other parts. Activities of PLD and LOX were induced by wounding, which higher activities of PLD and LOX was observed in navel end while membrane-associated Ca2+ content decreased. This result indicated that more mature tissue (navel) exhibited more sensitive in responses to wounding than younger tissues (middle and neck).We studied the variation of cucumber fruits antioxidant system to mechanical wounding. The rusults indicated that mechanical wounding could induced O2·- in cucumber fruits. During the storage of cucumber after wounding, the accumulation of H2O2 was provided to undergo a increasing-decreasing-increasing change, while the activity of SOD, POD, CAT, APX, and MDHAR could reach a maximu and then descent in the following hours. The activity variation of DHAR showed similar tendency to anti-oxidant enzymes with stable descend in the last storage period. Mechanical wounding could active the anti-oxidant enzymes in cucumber. Though the amplitude was much smaller, variation of GR activity in wounded cucumber was similar to H2O2 content. However, the wounded cucumber was always showing lower activity to control fruits. The contents of ASA and GSH were lower in wounded fruits than control, while the contents of DHA and GSSH were higher in wounded cucumber fruits than control fruits.We found that the order for contents of O2·- and H2O2 was navel end> middle> neck end. There was higher levels of O2·- and H2O2 in wounded cucumber fruits than control fruits. The activities of SOD, POD, CAT, MDHAR, APX, DHAR in navel end were higher than other parts of cucumber fruits. But activity of GR was different from other anti-oxidant enzymes, GR activity in navel end was the lowest in three parts of cucumber. Contents of ASA and GSH were as similar as GR in navel end, which was lower than control fruits. There were the highest contents of DHA and GSSG in navel than in middle and neck end, which induced lower GSH/GSSG in navel end. Therefore, the results suggest that response to mechanical wounding stress was maturity-dependent.Mechanical wounding could upregulate the gene expression of PLD, LOX, and some oxidation relative enzymes with time-effect. The expression levels of PLD and LOX in wounded fruits were significantly higher than control fruits. The expression of anti-oxidant enzymes such as SOD, POD, CAT, APX, MDHAR, and DHAR was higher in wounded cucumber fruits than control fruits in early hours of storge. However, the expression of these enzymes reduced sharply, and even lower than the control fruits during the later period of storge (8-10 h). The expression regulation of GR was accordance with other anti-oxidant enzymes though its expression was always lower than control fruits.The expressions of PLD, LOX, SOD, POD, CAT, APX, MDHAR, and DHAR in different cucumber parts were all higher than their control fruits following an order of fruit navel end> middle> neck end. The expression abundance of GR gene showed an opposite tendence to the above enzymes with an order of neck end> middle> fruit navel end. We finally concluded that the expression of anti-oxidant enzymes increased with the maturity of cucumber fruits.更多还原