一氧化氮调控采后桃果实线粒体膜通透性转换孔开放的分子机理

Mitochondrial membrane permeability transition pore (MPTP) opening is an important parameter indicting the integrity and functions of mitochondrial membrane, and has important effects on improving the qualities of postharvest fruits. This project investigates the regulation mechanism of nitric oxide (NO) on mitochondrial MPTP opening of peaches with modern analytical instruments and methods of structural chemistry and molecular biology. (1) By using surface plasmon resonance (SPR), two-dimensional electrophoresis and other technologies, the translation modifications of cyclophilin-D (CyP-D), adenine nucleotide translocator (ANT), voltage-dependent anion channel (VDAC) and phosphate carrier (PiC) by NO are detected, and the interactions between special His and Cys residues are studied. (2) The steric configurations of the complexes between NO and CyP-D, ANT, VDAC, PiC are simulated, and the relations between activities and the steric configurations are calculated in structural chemistry and biology. (3) By in vivo and in vitro experiments, the effects of NO on the activities and gene expressions of CyP-D, ANT, VDAC, PiC and HK Ⅱ are studied. (4) In fruits treated with NO and inhibitors of Ca2+ and reactive oxygen species (ROS), the indirect regulations by NO on MPTP opening through the pathways of Ca2+ and ROS are studied. With methods of chemistry and molecular biology, this project studies the relations between NO and mitochondrial MPTP opening, the molecular mechanisms that NO directly and indirectly regulates the MPTP opening in peaches. The results of this project provide evidences for disclosing the regulation mechanism of NO on the qualities of postharvest peach fruits.

线粒体膜通透性转运孔（MPTP）开放程度是反映线粒体膜完整和功能的重要指标，对保持采后果实品质意义重大。本项目将现代仪器分析方法和结构化学、分子生物学技术相结合，研究一氧化氮（NO）调控采后桃果实线粒体MPTP开放的机理。（1）利用表面等离子体共振、双向电泳等研究NO对亲环蛋白D（CyP-D）、腺苷酸转运蛋白（ANT）、电压依赖性阴离子通道（VDAC）及磷酸载体蛋白（PiC）的翻译后修饰及特定His和Cys残基相互作用；（2）从结构化学角度探讨NO与CyP-D、ANT、VDAC、PiC形成复合物空间构型与活性的关系；（3）活体和离体实验研究NO对CyP-D、ANT、VDAC、PiC、HK Ⅱ活性和基因表达的影响；（4）Ca2+、ROS阻断剂协同NO处理研究NO通过Ca2+、ROS途径对MPTP的间接调控。本项目研究NO直接和间接调控MPTP开放的机理，为NO调控采后桃果实品质提供理论依据。

线粒体膜通透性转换孔在线粒体控制的程序性死亡过程中起着至关重要的作用。本文以不同浓度的NO溶液和NO清除剂等处理的肥城桃果实为材料，检测其线粒体膜通透性相关指标，旨在研究NO调控采后果实线粒体膜通透性转换孔开放的分子机理，对于维持良好的果实品质，延长果实采后贮藏期具有重要的意义。.采用表面等离子体共振(SPR)法研究线粒体S-亚硝基化蛋白。对比PiC蛋白经亚硝基谷胱甘肽、适量还原剂DTT和对照的波数，结果表明NO可以将PiC上的巯基基团封闭，即发生亚硝基化作用，而还原性试剂如DTT可以还原亚硝基化产物。.采用分子生物学的方法，分析CyP-D、ANT、VDAC、PiC、HK Ⅱ这五种构成线粒体通透性转换孔（MPTP）的蛋白的全长cDNA，并进行蛋白表达、蛋白纯化与复性，获得基因全长和重组蛋白。采用酵母双杂技术检测蛋白间相互作用，研究不同浓度梯度的NO溶液对重组蛋白的荧光猝灭作用。并运用荧光定量PCR研究表明NO处理能够提高VDAC、CyP-D蛋白的基因表达量，但是会降低ANT基因的表达量，其中15 μmol L-1 NO处理的效果最为明显。.以体外制备脂质体囊泡人工构建磷脂双分子层膜模拟MPTP功能，并对其组分进行了探究，发现当MP和DPPC比例为1：1，浓度为0.1 mg mL-1时效果最好。.采用紫外分光光度法、荧光分光光度法研究NO对线粒体Ca2+介导的线粒体通透性转换的影响，并测定NO和ROS阻断剂（NAC）处理线粒体后的ROS含量、抗氧化酶活性、丙二醛（MDA）含量、线粒体膜电势和呼吸，以及NO处理后己糖激酶II（HK II）活性和基因表达量。研究表明，NO能够提高线粒体膜电势，抑制氧化应激下线粒体膜电势的降低，抑制线粒体中钙离子的外流，提高线粒体中ATP酶的活性，提高HKⅡ基因的表达量来维持激酶信号途径稳定，提高己糖激酶活性。.以上研究表明，NO处理能有效维持线粒体完整性和功能，降低贮藏期间果蔬线粒体损伤，保持良好的果实贮藏品质。