油菜hau CMS不育基因orf288的分子机理研究

Cytoplasmic male sterility (CMS) is a widespread natural phenomenon in higher plants. It provides an effective pollination control system in crop heterosis utilization, and has made great contribution to the increase of grain and oil production. However, there would be potential risks in the long-term use of the same male-sterile cytoplasm. On the other hand, special CMS materials can facilitate researches on cytoplasmic inheritance, pollen development and interactions between nuclear and cytoplasmic genes, and the clarification of molecular mechanisms for pollen sterility and fertility restoration in plant CMS can boost to aplly heterosis more efficiently..hau CMS is a newly discovered natural cytoplasmic male sterility in Brassica juncea by Professor Fu Tingdong in Huazhong Agricultural University. The pollen of hau CMS aborted completely and stably in several different environment. Therefore，This CMS system is better than the pol CMS, which was found also by Professor Fu in 1970's and has been used widely. We have screened out stable restoring materials for hau CMS..Previously, we have isolated and confirmed that orf288, located downstream of the atp6 gene and co-transcribed with this gene, is hau CMS-associated gene. Results also shown that orf288 itself has a targeting pre-sequence, and the ORF288 is a cytotoxic protein with transmembrane domains.In this project, we plan to investigate how the gene orf288 works. To analyze the effect of CMS-associated gene orf288 product on morphological characteristics and penetrability of mitochondrial membrane, the protoplasts of etiolated seedling root will be isolated and investigated. Yeast two-hybrid assay will be conducted to detect the interaction partners of ORF288. Two-dimensional gel electrophoresis and Brassica Gene Expression Microarray will be performed to analyze the differentially expressed genes between sterile and maintainer lines, and real-time PCR will be used to confirm these genes according to the results of genechip and yeast two-hybrid assay. Thus, we can clarify the function of the gene orf288 and provide helpful evidences to resolve the scientific issue: how the pollen is aborted in CMS in higher plants, and furthermore to make better use of CMS in plant heterosis utilization, and to provide guidances for discovering of novel CMS in crop breeding.

细胞质雄性不育是规模化利用作物杂种优势重要授粉控制系统之一，但长期利用同一不育胞质存在潜在的风险。hau CMS是傅廷栋教授新发现的天然芥菜型油菜细胞质雄性不育型，其不育性彻底、稳定，不受环境温度影响，优于现大面积应用的pol CMS，目前已获得稳定恢复材料。利用表达分析及克隆技术，我们分离了hau CMS不育基因orf288并对其进行了功能验证。本项目在此基础上，拟通过电镜观察和生化测定及基因芯片与蛋白质分析等，解析hau CMS不育基因orf288的调控网络以及其毒蛋白ORF288对线粒体膜形态结构与透性影响及作用方式，从而为阐明植物CMS花粉败育机理这一科学问题提供证据，并为更好地利用植物CMS杂种优势、创制新型植物CMS等提供指导。

细胞质雄性不育是油菜杂种优势利用的重要途径之一, 同时也是研究核质互作的重要实验材料。油菜hau CMS不育性彻底、稳定，具有广阔的应用潜力。本研究通过细胞学、遗传学和组学等的研究方法，对油菜hau CMS不育产生的原因进行了初步解析。细胞学研究发现，hau CMS不育败育发生在孢原细胞分化时期；不育系雄蕊中的线粒体空泡化，细胞肿胀，发育后期细胞被中央大液泡填充，细胞凋亡，最终导致雄蕊败育。线粒体基因组测序结果显示，hau CMS属于异源胞质，其不育系线粒体基因组发生了大量重组。表达分析结果显示，orf288在不育系的花药中高表达，在恢复材料的花药中表达量降低。不育基因orf288产物对大肠杆菌的生长有抑制作用，orf288截短实验显示，其毒性区域主要集中在它的三个跨膜结构域。遗传转化实验均证明orf288为hau CMS的不育基因，导致不育的核心区段主要在该蛋白的C端，表明育性可能与毒性无关。不育系和保持系差异表达分析结果表明，包括MADS box家族基因在内的很多参与早期雄蕊发育的基因在不育系中均下调表达，其他与氧化还原反应、细胞代谢和蛋白合成等路径相关基因在不育系中也下调表达。Western blot结果显示，仅不育系、恢复系中25KD处有明显特异性条带；转基因rfp+orf288（73-288aa）拟南芥中30KD处检测到特异性条带，而野生型中没有。这说明ORF288在蛋白水平也发挥作用。通过iTRAQ技术鉴定出不育系和保持系花蕾中186个差异表达蛋白，其中58个蛋白上调表达，128个蛋白下调表达；不育蛋白主要影响了蛋白酶体和细胞自噬途径，尤其是细胞自噬途径，导致蛋白降解功能失常，ROS大量积累，线粒体空泡化，碳水化合物和能量代谢失常，最终引起不育。通过构建诱饵载体288-BD和185-BD，对油菜花蕾酵母文库进行筛选，目前还在寻找与不育基因互作的蛋白。生理生化研究结果显示，不育基因的存在使得根尖中ROS升高，ATP含量降低，ATP酶活降低，膜电位降低，说明不育基因在根尖中表达，并在一定程度上影响了根尖的生长，但两者线粒体形态结构差别不大，表明不育基因对营养器官根的发育影响不大。