【作者】 雷磊；

【导师】 李友国；

【作者基本信息】 华中农业大学 ， 微生物学， 2014， 博士

【摘要】 根瘤菌在豆科植物根瘤内分化形成由宿主植物来源的膜系统包裹,能够固氮的细胞器,被称为共生体(symbiosome)。但是,对于这些植物来源的脂类是如何转运至共生体的,所知不多。本研究中,我们对紫云英根瘤中特异表达的转脂蛋白AsE246的功能及机制进行了研究。体外实验结果表明,重组AsE246蛋白具有脂质结合能力,能够和植物细胞膜上的脂质相结合。更重要的是,我们发现AsE246在体内可以与植物合成的脂质双半乳糖残基甘油二酯(DGDG)相结合。在洋葱下表皮中与红色荧光蛋白DsRed的融合蛋白表现出膜定位的特点。免疫荧光实验表明,AsE246基因编码产物从共生早期阶段,侵入线阶段就开始与共生体共定位。进一步的免疫电镜检测表明,AsE246基因编码产物定位于共生体膜。结果表明AsE246基因参与了共生体膜相关脂质的转运以及共生体形成过程。AsE246的超表达导致结瘤数增加,而RNAi则导致了结瘤数量减少,固氮酶活降低,DGDG含量降低,共生体发育异常,共生体膜皱缩,共生体周质空间增大,以及共生体内部积累聚p羟基丁酸(PHB)颗粒。对转基因植株的根瘤菌早期侵染时间的观察表明：AsE246通过影响侵入线的形成而进一步影响了根瘤的器官形成。综合以上结果可知,AsE246是共生体膜上所需脂质转运机制的重要组分,是豆科植物-根瘤菌共生关系建立中的必须基因。更多还原

【Abstract】 Rhizobia can form nitrogen-fixing organelles, called symbiosomes, inside the cells of legume root nodules, surrounded by a membrane of host plant origin. However, it is unknown how such plant-originating lipids are transported to the symbiosomes. In this study, we identified AsE246, a lipid transport protein that is specifically expressed in Astragalus sinicus root nodules.It was found that AsE246can bind lipids in vitro. More importantly, AsE246can bind the plant-synthesized membrane lipid digalactosyldiacylglycerol (DGDG) in vivo. In vitro fluorescent assays on the fatty acid1-pyrenedodecanoic acid demonstrated that the recombinant protein AsE246had the capacity to bind lipid. DsRed-AsE246fusion protein was found localized to the plasma membrane in onion epidermal cells. An immuno-fluorescence experiment showed that AsE246was colocalized with symbiosomes from an early stage of symbiosis between A. sinicus and Mesorhizobium huakuii7653R, even in the infection threads. Further immuno-electron microscopy (EM) result confirmed that AsE246was localized on the symbiosome membranes, indicating that AsE246participated in the transportation of related lipids and was involved in the biogenesis of the symbiosome membrane. Overexpression of AsE246resulted in increased numbers of root nodules. Knockdown expression of AsE246via RNAi resulted in the reduction of root nodule numbers, DGDG content and nitrogen fixation activity, and the abnormal development of symbiosomes, including the accumulation of poly-p-hydroxybutyrate, shrinkage in the symbiosome membrane and enlargement of the peribacteroid space. Observation of the symbiotic infection events suggested that AsE246affected nodule organogenesis associated with infection thread formation.Taken together, these results indicate that AsE246contributes to lipids transport to the symbiosome membrane, and this transport is required for effective legume-rhizobium symbiosis.更多还原