【作者】 李佩艳；

【导师】 郑小林；

【作者基本信息】 浙江工商大学 ， 食品科学， 2014， 博士

【摘要】 芒果、番茄属于冷敏型果实,在低温贮藏下易发生冷害,极大影响果实的贮藏期和品质。如何缓解低温贮藏下的冷害发生己成为冷敏型果实采后保鲜和品质调控亟待解决的关键问题。革酸是一种广泛存在于生物体的有机酸,草酸处理具有延缓果实采后成熟衰老进程,诱导提高果实的抗病性,降低果实褐变和冷害发生等效应。因此,草酸在采后果实贮藏保鲜中的应用研究已逐渐受到重视。为探讨草酸处理对冷敏型果实采后抗冷性的诱导作用及缓解冷害的机理,本文以芒果和番茄为材料,研究草酸处理对果实在低温胁迫下贮藏的冷害发生、品质变化、抗氧化代谢、脯氨酸代谢、能量代谢、差异表达基因等的影响。主要研究结果如下：1、低温胁迫下,芒果和番茄果实的冷害指数(CI)随贮藏时间的延长逐渐增加,贮藏末期草酸处理芒果和番茄的冷害指数分别比对照降低了25.32%和19.42%,这说明草酸处理能有效缓解果实采后的冷害。草酸处理显著抑制了芒果和番茄果实的呼吸速率和乙烯释放速率,延缓可滴定酸(TA)含量的下降；延缓芒果可溶性固形物(SSC)的上升,而对番茄SSC没有显著影响。另外,草酸处理延缓了芒果硬度的下降,但促进番茄后熟软化过程中硬度的下降。草酸处理延缓了采后果实成熟衰老进程,有助于维持低温贮藏下冷敏型果实的品质。2、低温胁迫下,草酸处理显著提高芒果和番茄果实的超氧化物歧化酶(SOD)、过氧化物酶(POD)、抗坏血酸过氧化物酶(APX)、谷胱甘肽还原酶(GR)、单脱氢抗坏血酸还原酶(MDHAR)、脱氢抗坏血酸还原酶(DHAR)酶活性,以及抗坏血酸(AsA)含量,从而显著提高芒果和番茄体内活性氧清除能力,维持较低的过氧化氢(H202)含量和超氧阴离子(02。-)生成速率,进而降低活性氧对细胞膜的损伤,提高细胞膜完整性,最终缓解果实在低温胁迫下的冷害发生。3、芒果中游离脯氨酸(Pro)含量在低温贮藏过程中呈逐渐下降趋势,但草酸处理显著提高了芒果Pro积累,提高吡咯啉-5-羧酸合成酶(P5CS)酶活性、抑制脯氨酸脱氢酶(PrDH)酶活性；番茄Pro含量在低温贮藏阶段逐渐下降,常温后熟阶段逐渐上升后下降,草酸处理显著提高了番茄Pro积累,提高P5CS和鸟氨酸转氨酶(OAT)酶活性、抑制PrDH酶活性。说明草酸处理对芒果和番茄的Pro代谢均有调控作用,而且Pro积累与提高P5CS、OAT酶活性、降低PrDH酶活性相关。同时,对番茄果实的P5CS、OAT、PrDH基因表达研究表明,贮藏过程中番茄P5CS、OAT、PrDH表达上升,而且草酸处理显著提高了番茄中P5CS、OAT基因表达,对PrDH基因表达没有显著影响。表明草酸处理通过调控番茄Pro生物合成的谷氨酸途径和鸟氨酸途径而使Pro积累,草酸处理调控芒果、番茄Pro生物合成代谢而积累Pro可能是其缓解冷敏型果实冷害发生的重要原因之一。4、低温胁迫下,草酸处理显著提高芒果ATP、ADP含量,显著抑制AMP含量上升和能荷的下降；草酸处理显著提高番茄常温后熟过程中ATP和ADP含量,但对番茄在低温胁迫下贮藏的ATP、ADP和AMP含量、以及能荷均没有显著影响。表明草酸处理显著提高芒果和番茄中能量物质的含量。草酸显著提高芒果和番茄中琥珀酸脱氢酶(SDH)、H+-ATPase、Ca2+-ATPase活性,说明草酸处理通过调节能量代谢及其相关酶活性而维持果实较高的能量水平,从而减轻低温胁迫下芒果和番茄的冷害。5、番茄红素不仅是番茄成熟的重要指标之一,也是番茄体内最主要的抗氧化物质之一,与番茄的抗逆性密切相关。草酸处理对番茄在低温胁迫下贮藏的番茄红素没有显著影响,但显著提高番茄从低温转入常温贮藏末期(20+9d和20+12d)的番茄红素含量。草酸处理显著提高番茄体内八氢番茄红素合成酶(PSY1)、<-胡萝卜素脱氢酶(ZDS)、八氢番茄红素脱氢酶(PDS)基因表达,从而导致番茄红素的积累。草酸处理提高番茄体内抗氧化能力等生理效应可能与其缓解低温胁迫下番茄的冷害相关。6、利用基因芯片技术筛选并得到草酸处理后贮藏20d、20+9d番茄果实全基因组表达谱中差异表达基因,结果表明20d番茄样本中共有1623条基因有差异表达,其中上调表达基因540条,下调表达基因1083条；20+9d番茄样本中共有1313条基因有差异表达,其中上调表达基因361条,下调表达基因952条。根据基因GO功能注释,草酸处理后20d以及20+9d番茄样本中的差异表达基因分子功能主要为结合活性、催化活性、裂解酶活性、氧化还原酶活性,参与防御反应、胁迫应答、代谢过程、氧化还原过程、磷酸化作用、细胞过程等生物学过程。GO富集分析结果表明,20d和20+9d番茄中差异表达基因分别有55个和47个功能聚类,富集基因条数较多的聚类参与胁迫应答、基础代谢过程、细胞代谢过程,此外还参与次级代谢过程。-PR蛋白、热激蛋白、病程相关蛋白P2、异柠檬酸裂合酶、蔗糖合成酶等12条基因在20d和20+9d番茄样本中均参与功能聚类；PSY1、PSY2、ZDS、几丁质酶、p-1,3-葡聚糖酶、琥珀酸脱氢酶、磷酸烯醇式丙酮酸羧化酶等只在20d或20+9d样本中有功能聚类。结合这些酶的差异表达倍数可得出,草酸处理明显调控了低温胁迫下番茄果实内抗性蛋白相关基因、能量代谢相关酶基因、番茄红素代谢酶基因的表达,推测草酸缓解番茄果实的冷害与抗性蛋白、能量代谢、番茄红素代谢等密切相关。更多还原

【Abstract】 Mango (Mangifera indica L.) and tomato (Solanum lycopersicum L.) fruit belong to chilling sensitive fruits, and harvested mango and tomato fruit are susceptible to chilling injury during storage at low temperature, which result in decrease in storage period and fruit quality extremely. Thus, it is very important to alleviate chilling injury in these fruit for their preservation and quality maintaining during storage at low temperature. Oxalic acid is an organic acid ubiquitously occurring in various organisms. Recently, pre-storage application of oxalic acid for postharvest fruit has received more attention, and it has been found that oxalic acid plays important roles such as delaying the ripening process, inducing disease resistance, and alleviating browning and chilling injury in postharvest fruit. In order to further understanding the effect of oxalic acid on alleviating the chilling injury in chilling-sensitive fruit, influences of oxalic acid in chilling injury, fruit quality, antioxidant capacity, proline acumulation, energy metabolism and expression of differential genes in mango and tomato fruit under chilling stress were investigated in this paper. The main results were as follows:(1) Chilling injury index in mango and tomato fruit gradually increased with increase in storage time under chilling stress. Compared to the control, the chilling injury index in both fruit decreased by25.32%and19.42%at the end of storage respectively, which showed oxalic acid treatment could effectively alleviate chilling injury in mango and tomato fruit during storage under chilling stress. Oxialic acid significantly inhibited respiration rate and ethylene production, retarded the decrease in firmnes and titratable acid (TA) and the increase in soluble solid content (SSC) in mango fruit, whereas it had no significant efficency in SSC and promoted the decrease in firmness in tomato fruit as transferring to room temperature from chilling stress. These effects of oxalic acid might collectively contribute to delaying the ripening and senescent process of postharvest fruit and thus to maintaining fruit quality for mango and tomato in storage under chilling stress.(2) Oxalic acid treatment obviously increased ascorbic acid (AsA) content and activities of superoxide disumutase (SOD), peroxidase (POD), ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR), which resulted in improvement on scavenging capacity for reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) and the production rate of superoxide anion (O2’") in mango and tomato fruit, and thus contributed to maintaining membrane integrity associated with alleviating the chilling injury in these fruit under chilling stress.(3) Proline content in mango fruit decreased during storage under chilling stress, but oxalic acid inhibited the decrease in proline content accompanied with increase in△1-pyrroline-5-carboxylate synthetase (P5CS) activity and decrease in proline dehydrogenase (PrDH) activity. Proline content in tomato fruit also decreased under chilling stress and increaseed thereafter decreased as transfering to room temperature. However, oxalic acid treatment gradually increased proline content accompanied with increase in activities of P5CS and ornithine-5-aminotransferase (OAT) and decrease in PrDH activity. Also, the gene expression of P5CS, OAT and PrDH in tomato fruit during storage was further studied. The data showed that the expression of P5CS, OAT and PrDH in tomato fruit were greatly enhanced during storage, and oxalic acid significantly improved the expression of P5CS and OAT except for PrDH, so that it seemed that proline accumulation in tomato fruit was associated with glutamic acid pathway and ornithine pathway, and oxalic acid might regulate proline accumulation for alleviating chilling injury in chilling sensitive fruit under chilling stress.(4) Oxalic acid treatment enhanced the content of ATP and ADP in mango fruit, and inhibited the increase in AMP and the decrease in energy charge during cold storage. In tomato fruit, neither the contents of ATP and ADP during cold storage nor AMP content and energy charge during the whole storage were affected by oxalic acid treatment, but the contents of ATP and ADP were enhanced as fruit transferred to room temperature.. Furthermore, oxalic acid treatment increased activities of succinodehydrogenase (SDH), H+-ATPase and Ca2+-ATPase both in mango and tomato fruit, it was suggested that oxalic acid treatment could regulate energy metabolism for alleviating chilling injury in mango and tomato fruit under chilling stress.(5) Lycopene, as an important indicator for ripening and a primary antioxidant in tomato fruit, is closely related with stress resistance for fruit.. Oxalic acid treatment did not affect lycopene in tomato fruit during cold storage, but resulted in higher lycopene content at the latter days (20+9d and20+12d) as fruit were removed to room temperature. Also, oxalic acid treatment enhanced the gene expressions of phytoene synthasel (PSY1),ζ-carotene desaturase (ZDS) and phytoene desaturase (PDS). Thus, it was suggested that effect of oxalic acid on the accumulation of lycopene might also contribute to improving antioxidant ability in tomato fruit, and in turn for alleviating chilling injury in tomato fruit under chilling stress.(6) For obtaining an overall view on transcript modification and finding differentially expressed genes in tomato fruit with oxalic acid treatment during storage under chilling stress, a microarrary analysis was carried out by using Affymetrix tomato genechip arrays. As compared to control fruit,1623genes expressed more than two folds in oxalic acid treated fruit in storage under chilling stress for20days, in which540and1083genes were up-regulated and down-regulated, respectively. As fruit were removed to room temperature for9days (20+9d),1313genes still expressed more than two folds in treated fruit, in which361and952genes were up-regulated and down-regulated, respectively.The differentially expressed genes were analyzed by GO annotation. The differentially expressed genes in oxalic acid treated tomato fruit mainly involved in stress response, defense response, metabolic process, oxidation-reduction process, phosphorylation and cellular process, which were associated to binding, catalytic activity, lyase activity and peroxidase activity. Also, GO enrichment analysis showed that the differentially expressed genes in20d and20+9d tomato sample had55and47annotation cluster, respectively. Annotation cluster with more enrichment genes mainly participated in stress response, primary metabolism, cell metabolism process and secondary metabolism.12genes, including pathogenesis related protein (PR protein), heat shock protein, PR-P2pathogenesis-related proteins, isocitrate lyase and sucrose synthetase, participated in annotation cluster both in20d and20+9d tomato sample, while PSY1, PSY2, ZDS, chitinase, Beta-1,3-glucanase, succinodehydrogenase and phosphoenolpyruvate carboxylase participated in annotation cluster only in20d or20+9d sample. Based on the fold change of those differentially expressed genes, it was suggested that oxalic acid regulated the protein expressions including resistance protein and key enzymes involed in energy metabolism and lycopene metabolism, which might contribute to alleviating chilling injury in tomato fruit.更多还原