过氧化氢介导的细胞程序性死亡在竹笋木质化进程中的作用

Lignification of harvested fruits and vegetables during storage is an important scientific issue in postharvest research area. There is by far no domestic report demonstrating the relationship between programmed cell death (PCD) and lignification in postharvest fruits and vegetables. Based on the result on the bamboo shoot, new technologies such as electronic microscopy, western blotting and terminal-deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) were used in the project. Through the study of lignin metabolism and hydrogen peroxide metabolism in bamboo shoots, the relationship between lignification and PCD, induced by hydrogen peroxide, were investigated from three different levels: cell wall ultrastructure, protein western blotting and the transformation of DNA, with the focus on PCD related cell morphology, biochemistry and molecule biology. This project also discussed the association between hydrogen peroxide and protoplast degradation, nuclear DNA fragmentation and lignin deposition. The influence of hydrogen peroxide on the launch, occurrence and development of lignifications PCD was discussed and the possible modulation pattern was revealed. Starting from a new aspect, the study tried to offer a new explanation of the lignification mechanism in bamboo shoots, emphasizing more on molecule biology and cytobiology rather than physiology as most current researches did. The attempt would make a meaningful contribution to the development of plant PCD theory and the plant lignification mechanism, as well a new research concentration in postharvest fruits and vegetables.

果蔬采后木质化机理和调控是当前深受许多研究者所关注的一个重要科学问题，国内对细胞程序性死亡（PCD）与采后果蔬木质化的研究还未见有报道。本课题在前期研究成果基础上，利用电子显微、蛋白质印迹和核酸原位末端标记标记等新技术，从竹笋木质素代谢入手，以H2O2代谢为纽带，以竹笋贮藏期间PCD过程相关的形态学、生化学和分子生物学特征为核心，从细胞超微结构、蛋白质表达和DNA变化等三个不同层次研究 H2O2诱导的竹笋PCD与木质化的关系，探讨H2O2在竹笋原生质体降解、细胞核DNA断裂和细胞壁木质素沉积间的联系及其机制，揭示H2O2在竹笋与木质化PCD的启动、发生、发展中的作用和可能调控模式。目前对采后果蔬木质化的研究主要从生理学水平开展，本课题则侧重于分子生物学和细胞生物学水平的研究，从一个全新的角度来阐述竹笋木质化机理。这对丰富植物PCD理论，完善植物木质化机理和提高采后果蔬品质具有重要科学意义。

本项目从竹笋木质素代谢入手，以H2O2代谢和组织定位为纽带，以竹笋贮藏期间木质化过程相关的细胞形态、生理生化学和分子生物学特征为核心，探讨在20℃下H2O2诱导的竹笋木质化进程。研究发现，外源H2O2处理可以促进竹笋木质素的合成，而DPI处理则抑制了竹笋的木质化进程。外源H2O2处理还提高NADPH氧化酶活性和和内源H2O2含量，而DPI处理抑制NADPH氧化酶活性和和显著降低内源H2O2含量。从细胞形态学的角度研究发现，DPI处理竹笋样品的细胞壁较薄，而H2O2处理的竹笋样品在电镜下观察到细胞液泡解体，细胞壁明显加厚。DNA Ladder图谱表明，从竹笋顶部开始，DNA的片段化逐渐明显，DPI处理可抑制DNA的断裂，而H2O2处理则促进了竹笋组织的DNA的断裂。采后竹笋DNase和RNase活性增加，外源H2O2处理显著提高了DNase和RNase活性，而DPI处理抑制DNase和RNase活性。采后竹笋的底部可以明显的观察到TUNEL 检测反应呈阳性，显示此时的纤维细胞核DNA 发生了片段化，能检测到类Caspase-3酶活性，通过Western blotting检测技术，发现了一个分子量为32KD的类caspase-3蛋白。这些研究结果表明H2O2处理促进采后竹笋木质化和PCD的进程，而DPI处理则可以以延缓这一进程。本研究提出了H2O2在竹笋PCD 与木质化间的可能调控作用及模式，明确采后竹笋贮藏期间H2O2诱导的竹笋PCD与木质化的关系及作用机制。本研究发表论文20篇，其中在SCI刊物上收录19篇，EI收录1篇；授权国家发明专利3项；培养博士后1名，博士生3名，硕士研究生3名。