梨自花花粉管NADPH氧化酶介导线粒体功能紊乱的机制研究

Mitochondrial dysfunction is closely associated with the cessation of pear self-incompatible pollen tube growth. Thus it is very important to study the mechanism(s) of the mitochondrial dysfunction for the resolution of reproduction isolation resulted from self-incompatibility. Although NADPH oxidase plays significant roles during pollen tube growth, its effects on regulating mitochondrial dysfunction remain largely unknown in self-incompatible pollen tube. Therefore, our project is intended to use pear cultivars ‘Cuiguan’ and ‘Huanghua’ as plant materials; transient gene silence, immunofluorescence label assay, laser confocal scanning microscope and other techniques were applied in the project. The effects of NADPH oxidase alternations on mitochondrial movement will be revealed and the potential roles of NADPH oxidase during mitochondrial dysfunction will be further clarified in pear self-incompatible pollen tube. The project’s aim is to elucidate the mechanism underlying mitochondrial dysfunction mediated by NADPH oxidase in pear self-incompatible pollen tube. This will improve the mechanism of pear self-incompatibility, provide theoretical basis for breeding of self-compatible new pear cultivars.

梨自花花粉管生长停止与线粒体功能紊乱密切相关，因此研究自花花粉管线粒体功能紊乱的发生机制对解决自交不亲和性导致的生殖隔离问题具有重要意义。虽然花粉管NADPH氧化酶对其正常生长发育不可或缺，但是自花花粉管NADPH氧化酶在线粒体功能紊乱中的调控作用尚不清楚。本项目拟以砂梨品种‘翠冠’和‘黄花’为研究材料，综合应用基因瞬时沉默、免疫荧光探针标记、激光共聚焦显微镜等研究技术和手段，探讨梨自花花粉管NADPH氧化酶变化对线粒体运动的影响及其在线粒体功能紊乱中的调控作用。旨在阐明梨自花花粉管NADPH氧化酶介导线粒体功能紊乱的机制，进一步完善梨自交不亲和性机理，为自交亲和性梨新品种的选育提供理论依据。

梨是基于S-RNase的配子体型自交不亲和性果树，前期研究发现梨自花花粉管线粒体结构发生损伤及细胞色素c释放，并具有程序性细胞死亡的典型特征。同时，花粉管NADPH氧化酶对其正常生长发育不可或缺，但是梨自花花粉管NADPH氧化酶在线粒体功能紊乱中的调控作用还不清楚。基于此，本项目围绕梨自花花粉管线粒体功能紊乱与NADPH氧化酶的关系开展相关研究。研究结果表明克隆得到两个梨花粉管NADPH氧化酶基因（Prrbohs）为花粉管中特异表达基因，梨自花花粉管Prrbohs基因表达显著（P<0.01）降低，NADPH氧化酶活性显著（P<0.01）下降，并由此导致花粉管尖端ROS浓度梯度遭到破坏。进一步研究了梨花粉管NADPH氧化酶变化对微丝骨架形态的影响，结果表明梨花粉管NADPH氧化酶活性降低诱导了微丝正常形态的破坏。通过分析梨自花花粉管NADPH氧化酶变化对线粒体分布的作用特点，发现自花花粉管亚尖端线粒体富集分布遭到特异性破坏。梨花粉管NADPH氧化酶基因瞬时沉默实验结果显示梨花粉管线粒体正常的分布状态受到明显干扰，同时微丝解聚剂Lat-B也扰乱了梨花粉管线粒体正常分布，这说明梨自花花粉管NADPH氧化酶变化诱导微丝骨架解聚介导了线粒体正常动态分布的紊乱。通过对梨花粉管NADPH氧化酶变化对线粒体膜功能的影响分析，结果表明梨花粉管NADPH氧化酶活性降低导致了线粒体膜电位显著（P<0.01）下降，而线粒体膜电位降低与花粉管呼吸氧消耗以及胞内ATP含量下降密切相关。同时结果显示梨自花花粉管线粒体膜通透性转换孔（membrane permeability transition pore，mPTP）遭到了特异性破坏，其中自花花粉管NADPH氧化酶的变化是线粒体mPTP紊乱的主要诱因之一。本研究明确了梨自花花粉管NADPH氧化酶变化诱导微丝骨架解聚介导了线粒体正常动态分布的破坏，由此进一步导致花粉管呼吸代谢降低，线粒体mPTP开放及膜电位下降等功能性紊乱。本研究结果完善了蔷薇科果树自交不亲和性机理，为自交亲和性梨新品种的选育提供了相关的理论依据。