基于糖代谢与活性氧关系对采后番木瓜果实异常冷害行为机制研究

Chilling injury (CI) is a kind of common physiological disorder in postharvest fruits and vegetables. Generally, within the temperature range during which chilling injury occurs, the lower the storage temperature, the more serious the degree of chilling injury. However, in papaya fruit, the more lower temperature of the chilling injury, the CI symptoms of fruit is relatively lighter. A large number of studies have shown that reactive oxygen metabolism is closely related to cell membrane damage and ultimately leads to chilling injury. In recent years, it has been found that there is a certain correlation between sugar metabolic and reactive oxygen species (ROS). Moreover, sugar is the main constituent and respiratory substrate of postharvest papaya fruit. Therefore, this project is aimed at the papaya fruit under different chilling temperatures, with the fruit tissue changes as the entry point. Research focus on the relationship between sugar metabolism and ROS to study the effects of different chilling temperatures on fruit saccharides and their metabolism pathway（saccharides transformation and respiratory metabolism pathway）, ROS and the enzymes which could scaveng ROS. Furthermore, the effects of different chilling temperature on carbohydrate metabolism in papaya fruit were researched from the change of gene expression level of key enzymes of sugar metabolism. Thereby, the relationship between sugar metabolism and reactive oxygen species at different chilling temperatures and the regulation mechanism of chilling temperature on sugar metabolism will be finally clarified, and the mechanism of sugar metabolism in the abnormal cold injury of papaya fruit will be revealed. The biological mechanism of chilling injury of postharvest fruit will be further enriched.

冷害是果蔬采后常见的一种生理性病害。一般而言，在诱发冷害温度范围内，温度越低，冷害程度越严重。但在番木瓜果实中，在更低冷害温度下，果实冷害症状反而较轻。大量研究已表明活性氧代谢与细胞膜破坏密切相关，并最终导致冷害发生。近年发现，糖及其代谢途径变化及相关酶与活性氧之间具有一定关联性。而且，糖是采后番木瓜果实中主要组织成分和呼吸基质。因此，本项目以不同冷藏温度下番木瓜果实为对象，从果实细胞组织结构变化为切入点，以糖代谢与果实组织中活性氧关系为核心，研究不同冷藏温度对果实糖分、糖代谢途径（糖分的转化和呼吸代谢途径）、活性氧及其清除酶系的影响；并进一步从糖代谢关键酶基因表达水平变化探讨不同冷害温度对番木瓜果实糖代谢的影响作用。从而，最终阐明不同冷藏温度下糖代谢与活性氧之间的关系以及冷藏温度对糖代谢的调控机制，揭示糖代谢在番木瓜果实异常冷害行为中的作用机理，进一步丰富采后果实冷害发生的生物学机制。

针对番木瓜果实在更低的冷温下反而较更高冷温下冷害症状较轻的异常冷害行为，以1和6℃冷温贮藏的番木瓜果实为对象，重点从糖代谢与活性氧之间关系展开研究。结果表明1℃较长时间保持了果实表皮组织细胞完整性，减缓了果实中活性氧生成。究其原因，一方面，1℃冷藏诱导了果实中CpSOD、CpCAT、CpAPX和CpGR基因表达量上调，使其保持了较高活性氧清除酶活性和GSH/AsA含量；另一方面，糖分及其代谢也与活性氧间有关。1℃使果实中淀粉和果糖在整个贮藏中均显著地高于6℃果实，而葡萄糖和蔗糖主要在中后期维持了较高水平，H2O2和O2-·与淀粉、葡萄糖和果糖含量均呈极显著负相关（P < 0.01）。而1℃果实之所以能维持较高糖分，从糖分间转化角度看，1 ℃提高了果实中CpSPS基因表达水平，抑制了CpAI基因表达，与此同时，果实保持了较低的淀粉酶、酸性转化酶和较高蔗糖合成酶分解方向活性和较高的中性转化酶、蔗糖合成酶合成方向和磷酸蔗糖合成酶活性。前者有助于延缓葡萄糖和果糖下降，后者有助于维持较高蔗糖水平；从呼吸代谢角度看，1℃冷藏显著地降低了果实总呼吸速率，推迟了呼吸高峰出现，并抑制了果实中后期EMP-TCA呼吸代谢。但贮藏中后期显著地促进了CpG6PDH和Cp6PGDH基因的表达，提高了磷酸戊糖代谢途径（PPP）关键酶活性，刺激了PPP途径，促进了果实中后期组织中NADP+和NADPH含量的增加。同时，贮藏中后期，O2·-和H2O2均与PPP途径及其产物NADPH含量呈相反关系；从电子传递链环节来看，1 ℃冷藏显著地抑制了果实中CpCCO的表达而诱导了CpAOX表达量上调，从而抑制了果实中下细胞色素氧化酶（CCO）活性，促进了贮藏后期交替氧化酶（AOX）活性上升。同时提高了对AOX具有重要调控作用的泛醌含量及其还原态水平和丙酮酸含量。其结果抑制了果实中细胞色素途径(CP)，刺激了中后期抗氰呼吸途径（AP），提高了AP比重。O2-·和H2O2均与AP间则呈现出一定负相关性。该研究证明了番木瓜果实中糖分及其代谢与活性氧之间同样密切相关，并在其异常冷害行为中扮演了重要角色，丰富了采后果实冷害发生的生物学机制。