硫化氢调控活性氧代谢抑制鲜切苹果褐变机理的研究

Enzymatic browning of fresh cut fruits and vegetables was closely related to the ROS metabolism. The hydrogen sulfide (H2S) was recognized as a new gaseous signal molecule with the ability to inhibit enzymatic browning of fresh cut apples by regulating ROS metabolism, however, the underlying mechanism is still obscure. In this project, the technologies and methods of molecular biology, biochemistry, cell biology and transcriptomics will be used to study the mechanism of H2S regultes ROS metabolism to inhibit enzymatic browning of fresh-cut apples. The real time changes and subcellular localization of ROS of fresh cut apples after H2S donor treatment will be monitored. Meanwhile, genic expression and enzymic activity from ROS biosynthesis and scavenging system will be investigated to determine the regulating effect of H2S on ROS metabolism. Meanwhile, the functional genes related to ROS regulation will be identified by analyzing the transcriptome of fresh cut apples after H2S treatment in order to explore the molecular mechanism of H2S regulating the ROS metabolism. Furthermore, in order to reveal the relationship between the enzymatic browning and ROS metabolism, a prediction model for browning based on ROS metabolism will be developed. This study will demonstrate the mechanism of H2S inhibits enzymatic browning of fresh-cut apples through regulating ROS metabolism at the biochemical and molecular level. In conclusion, our study will provide a theoretical basis for enriching the biological theory and developing preservation technology of fresh-cut fruits and vegetables.

鲜切果蔬酶促褐变与活性氧（ROS）代谢有着密切的关系，新型气体信号分子硫化氢（H2S）可以通过调控ROS代谢抑制鲜切苹果的酶促褐变，但其调控机理尚不清楚。本项目以鲜切苹果为研究对象，采用外源H2S供体处理，综合分子生物学、生物化学、细胞生物学和转录组学等多学科方法，动态监控低温贮藏过程中鲜切苹果ROS含量变化及亚细胞定位，分析H2S处理对ROS生物合成及清除系统相关酶基因表达和酶活性变化的调控作用，进一步通过对H2S处理鲜切苹果的转录组分析，深度挖掘H2S调控ROS代谢的功能基因，从而解析H2S调控ROS代谢的分子机制。此外，结合鲜切苹果贮藏期间的表观褐变进程，建立基于ROS代谢的褐变预测模型，明确 H2S调控鲜切苹果ROS代谢途径与酶促褐变的相关性，从而在生化和分子水平上解析H2S调控ROS代谢抑制鲜切苹果褐变的机理，为丰富鲜切果蔬生物学基础理论及开发鲜切果蔬贮藏保鲜技术提供理论依据。

酶促褐变是鲜切苹果加工与贮藏保鲜过程中面临的主要问题之一，有效控制鲜切苹果的褐变是当前研究的热点问题。新型气体信号分子硫化氢（H2S）可以有效抑制鲜切苹果的酶促褐变，但其调控机理尚不清楚。本项目从“H2S处理→调控ROS代谢→抑制褐变”的角度，通过动态监控鲜切苹果在贮藏期间ROS产生规律，ROS代谢途径相关酶活性以及抗氧化物质含量，同时利用RNA-Seq技术对H2S处理后鲜切苹果的转录组进行测定和分析，深入分析H2S调控ROS代谢抑制鲜切苹果褐变的分子机制。结果显示H2S处理显著降低了鲜切苹果在贮藏期间ROS的积累，提高了其超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、谷胱甘肽还原酶（GR）等抗氧化酶的活性以及Vc和谷胱甘肽（GSH）等抗氧化物质的含量，从而提升了鲜切苹果的ROS代谢能力；同时，H2S熏蒸处理提高苯丙氨酸解氨酶（PAL）活性，不同程度提高了绿原酸、单宁酸、表儿茶素和香草酸等多酚类物质的含量；相关性分析表明鲜切苹果的褐变程度与ROS含量呈显著正相关，与SOD、GR和PAL活性、GSH、VC、槲皮素、绿原酸、表儿茶素及香草酸含量呈显著负相关，证明H2S通过提高鲜切苹果抗氧化防御能力，维持ROS代谢平衡，从而减轻ROS对组织产生的氧化损伤，进而抑制其褐变；RNA-Seq分析结果显示，鲜切苹果经过H2S熏蒸处理后，多数参与抗氧化代谢和能量代谢的基因显著上调，而编码PPO、和膜脂降解相关酶，例如脂氧合酶和磷脂酶D显著下调，进一步证实H2S在转录水平上调节鲜切苹果的ROS代谢、能量代谢和脂类代谢，从而保持膜的完整性，延缓PPO与酚类物质的反应，进而控制其褐变的发生。本研究为丰富鲜切果蔬生物学基础理论及开发鲜切果蔬贮藏保鲜技术提供理论和实验依据。