基于差异蛋白质组学的冷藏南果梨果皮褐变机制研究

Nanguo pear (Pyres ussuriensis Maxim) is a special local fruit in north China, and its mature fruit is excellent in quality for delicate pulp, rich juice, mellow and tasty. It is difficult to store Nanguo pear at ambient temperature for the problem of softening and deterioration in about half a month after picking. By using refrigeration technology developed in our laboratory, the storage duration can be extended to 6 months. However, the long-term refrigerated fruit prone to pericarp browning after being removed to room temperature condition, which influences its comercial value seriously. It was noted in our preliminary study that process cooling and intermittent heating could effectively alleviate the pericarp browning of the refrigerated fruit. In this program, by using three kinds of treated fuits as experiment materials including process cooling, intermittent heating and direct cooling, we will separate and screen differentially expressed proteins which link to the pericarp browning by the two-dimensional gel electrophoresis and mass spectrometry analysis, classify the functions of the proteins identified according to the bioinformatics tool for functional classification Mapman, predict their metabolic pathway that may be involved in. At the same time, observe and determine the alteration in structure and function of membrane system, lipid composition of membrane and its peroxidation. Integrating the results of proteomics with those coming from subcellular structure and physiological and metabolic data we will excavate the metabolic pathways associated with the pericarp browning of the fruit, which will allow us to elucidate at the mechanism of the pericarp browning of Nanguo pear. The results of this program will lay the foundation for developing browning inhibiting technology for Nanguo pear.

南果梨为我国北方秋子梨系的特产水果，果肉细腻多汁、醇香味美。采收后的南果梨在室温下极不耐贮，半月左右果实即变软腐烂。本实验室研究开发的南果梨冷藏保鲜技术，可使其贮藏期延至6个月，但经长期冷藏的南果梨转入室温后果皮极易发生褐变。本实验室前期研究发现，程序降温和间歇升温可以有效地缓解冷藏南果梨果皮的褐变。本项目拟采用常规的快速降温、以及程序降温和间歇升温3种冷藏方法贮藏南果梨，在果实出库后的常温货架期，选取褐变与非褐变果实，利用双向电泳技术结合质谱分析，分离和筛选与果皮褐变相关的蛋白质，利用Mapman软件对筛选出的蛋白进行功能分类，预测其可能参与的代谢途径。同时，分析测定冷藏过程中果皮细胞膜系统的结构与功能、膜脂组分及其过氧化产物的变化。综合蛋白质组学与亚细胞结构、生理和代谢的研究结果，深入挖掘与果皮褐变相关的代谢途径，揭示冷藏南果梨果皮褐变的机制，为南果梨防褐保鲜技术的开发奠定基础。

冷藏南果梨转入常温货架期后果皮很容易发生褐变，长期冷藏的果实甚至在果实尚未后熟软化即表现出褐变症状，严重影响了冷藏南果梨的商品品质。本项目利用iTRAQ分析共鉴定冷藏南果梨果皮褐变差异表达蛋白质181个，其中，95种蛋白下调表达，86种蛋白上调表达。GO分析结果表明，差异蛋白主要参与到生物过程中的“细胞过程”，“应激反应”和“细胞组分组织或合成”；细胞组分分类中的“膜”；分子功能分类中的“结合”与“催化活性”。KEGG分析结果表明，“碳代谢”，“氨基酸的生物合成”和“核糖体合成”是差异蛋白质最富集的途径。.长期冷藏的南果梨出库后常温货架期间，果皮中ATP含量和EC值都显著下降，氧化磷酸化代谢途径中的NADH脱氢酶基因（NDH）、无机焦磷酸酶基因（VPP）和ATP合成酶（ATPase）基因的表达量都发生下调；丙酮酸代谢途径中乙酰辅酶A羧化酶基因（ACC）和苹果酸脱氢酶基因（MDH）显著发生下调表达；谷胱甘肽代谢途径中谷胱甘肽过氧化物酶基因（GPX）表达量发生上调表达。说明了能量代谢和膜质代谢与南果梨果皮褐变是密切相关的。外源ATP处理进一步证明了能量代谢和膜质代谢水平与南果梨果皮褐变的关系.阶段性降温（LTC）和间歇式升温（IW）两种处理都可以延迟冷藏南果梨在常温货架期间果皮褐变现象的发生，并显著降低了果皮的褐变指数，显著提高了果皮中ATP含量和EC值。两种处理南果梨果皮NDH、VPP和ATPase的基因均上调表达，ATP含量和EC值有所提高，说明这两种处理能使细胞维持较高的能量水平，以维持细胞的正常代谢。同时，两种处理使磷脂酶D基因（PLD）表达水平发生下调，MDA含量降低，说明果实的膜脂过氧化程度放缓，细胞的区室化结构分布得到更好的维护。此外，两种处理南果梨果皮ACC和MDH基因发生上调表达，反映出果实的脂肪酸合成代谢途径趋于稳定，维护了细胞膜的合成和稳定。两种处理还缓解了GPX基因表达的上调水平，可以推断两种处理促进了果皮细胞的正常生理代谢过程，提高了果皮细胞的防御系统活性。