膜结合态多酚氧化酶对苹果CO2伤害褐变的调控机制研究

Browning of apple flesh which caused by the CO2 injury is one of the main problems of postharvest fresh-keeping. Browning is mainly attributed to the action of polyphenol oxidase (PPO) which exist two forms: membrane-bound (mPPO) and soluble (sPPO). About 90 % of PPO is mPPO in apple. But the regulatory mechanism of mPPO in apple browning caused by CO2 injury is unclear now. Our research on bioinformatics of mPPO shows that it is related to apple browning with a higher activity than that of sPPO and also involves in response to stress caused by CO2 injury. The regional distribution, dynamic, the conversion relationship with sPPO and gene expression changes of mPPO in browning caused by CO2 injury will be studied by means of biochemistry, enzymology and molecular biology to reveal the regulatory mechanism of mPPO in apple browning induced by CO2 injury. In order to further understand the mechanism of browning induced by CO2 injury, physiological metabolism, cell ultrastructure and the defense response of the antioxidant system also will be analyzed. The results of this study will gain a better understanding of the process of the physiological and biochemical metabolism of apple induced by CO2 injury. And it is of great significance in building science and technology theory of apple fresh-keeping and as well as in realizing industrialization of fruits and vegetables preservation.

苹果CO2伤害褐变是采后贮藏保鲜的主要问题之一。苹果中多酚氧化酶(PPO)分为游离态(sPPO)和结合态(mPPO)两种存在形式，其中mPPO约占90%，但目前mPPO在苹果CO2伤害褐变中的作用及其调控机制尚不明确。申请人前期研究发现mPPO参与苹果褐变，且催化活性显著高于sPPO，同时mPPO与苹果CO2伤害胁迫响应相关。本项目拟利用生化、酶学及分子生物学技术，动态描述mPPO在苹果CO2伤害褐变中的活性变化、区域分布、与sPPO的形态转化和基因表达差异，结合苹果CO2伤害褐变中果肉细胞生理代谢、细胞超微结构的变化及抗氧化酶系统的防御反应，全面了解苹果CO2伤害褐变中基因表达、物质代谢的差异，揭示苹果mPPO对CO2伤害褐变的调控机制。研究结果对揭示贮藏过程中苹果CO2伤害的生理生化代谢，构建苹果贮藏保鲜的科学技术理论体系及实现新鲜健康的果蔬保鲜产业化具有重要意义。

苹果二氧化碳（CO2）伤害褐变是一个涉及生理、生化、细胞结构、细胞代谢（包括化合物代谢、能量代谢及脂质代谢）等多个方面的变化，该项目建立了CO2胁迫处理后结合态多酚氧化酶（mPPO）和可溶态多酚氧化酶（sPPO）的动力学模型，明确了mPPO及sPPO在不同苹果品种CO2耐受性差异中的作用；揭示了富士苹果mPPO、sPPO在CO2伤害褐变中的区域性分布及活性变化的动态趋势；分析明确了苹果CO2伤害褐变发生过程中保护酶系统活性变化、细胞膜电导率变化及细胞组织结构变化之间的关系；初步阐明了苹果CO2伤害褐变发生的过程及部分机制。细胞区域化结构破坏导致的酶褐变并不是引起果肉组织褐变的唯一途径，苹果CO2伤害褐变呈现阶段性变化。根据我们实验结果推测CO2伤害褐变分为三个进程：第一进程（0-7 d，未褐变，主要是生理生化变化，酶活的表达差异，调节）；第二进程（7 d-14 d，还未出现细胞解体，但已经出现轻微褐变，此时的褐变可能主要是氨基酸及Vc参与的）；第三进程（14 d以后，细胞结构破坏，大面积出现褐变，此时发生的主要是sPPO与多酚的酶促褐变。）。研究结果对揭示贮藏过程中苹果CO2伤害的生理生化代谢，构建苹果贮藏保鲜的科学技术理论体系及实现新鲜健康的果蔬保鲜产业化具有重要意义。