耐低温根瘤菌的低温适应分子机理研究

Nitrogen fixed by rhizobium-legume symbiotic system is sustainable nitrogen source of agriculture. However,temperate agro-ecosystems are characterized by periodic low temperatures that subject both legumes and rhizobia to cold temperature induced stress. Many studies have shown that sub-optimal temperatures reduce the growth rate and the competitiveness of mesophilic rhizobia for nodulation, repress the expression of secretion system genes and release of nod factors,delay root infection and inhibit nitrogenase activity. Therefore, it is of great significance to investigate the cold-adaptation mechanisms of psychrotrophic rhizobia which could form efficient nitrogen-fixing symbiotic system with legumes at low temperatures. However, our knowledge in this field is very limited, especially abouth the roles membrane proteins playing in the cold-tolerance and the rhizobium-legume interaction procedure in cold adaptation. We isolated a psychrotrophic rhizobium strain YM-1 from Hulun Buir grassland soil. It grows well at low temperatures and could form efficient nitrogen-fixing symbiotic system with host legume under cold stress. In this study, we will sequence the genome of YM-1 and screen genes and proteins involved in the cold adaptation of YM-1 by utilize the technologies of comparative genomics, outer-inner membranes seperation and proteomics, and to verify their function in cold-adaptation by gene mutation and complement experiments;by this way, we can understand the molecular mechanism of cold adaptation in YM-1 from a global view to a specific way;the cold-acclimation proteins expressed during the symbiotic procedure will also be screened by combining compartmentalized incubation method;quorum sensing will also be explored to find out if it is involved in the cold adaptation during the early stage of symbiosis. The results of this project will give a comprehensive insight into the cold-adaptation mechanisms of YM-1 and provide solid theoretical bases for further research in this area.

根瘤菌-豆科植物共生固氮体系是可持续的农业氮素来源，但目前利用较多的中温根瘤菌对低温胁迫极为敏感，早春低温会严重影响其共生固氮效率，导致豆科作物大幅度减产。因此，筛选和利用低温下能与宿主高效结瘤固氮的耐低温根瘤菌和研究其低温适应机理具有重要意义，但相关研究却很有限。本项目组从温带草原土壤中分离了一株能在低温下快速生长并与宿主形成高效共生固氮体系的根瘤菌YM-1。本研究将以YM-1为模式菌株，通过基因组测序分析、比较基因组学、蛋白质组学结合细胞内外膜分离技术鉴定YM-1低温适应相关基因，然后通过基因突变和互补实验验证这些基因在低温适应中的作用，从"面"到"点"了解YM-1的低温适应分子机理；同时结合分室培养箱培养法筛选和鉴定低温下YM-1与宿主互作过程中表达的低温适应蛋白；并探讨群体感应是否与低温适应相关。本研究的结果将为根瘤菌低温适应分子机理分析提供全面的信息，为更深入的研究提供理论基础。

耐低温菌，即在低温条件下能快速生长，但最适生长温度超过15℃的细菌，它们在自然环境中广泛分布。能在低温条件下生长良好，意味着在一些周期低温温度地区，它们一年中进行活跃的生命代谢活动时间长，可能给予它们其它中温微生物所不具备的生态优势，从而发挥重要的生态功能，。但是关于其生态功能未见研究报道。本项目采用组学、生理生化试验和分子生物学技术，研究了耐低温菌YM-1的低温适应机制和生态学意义。具体开展的研究包括：（1）测定了YM-1的基因组并对测定的数据结合蛋白组学数据进行了初步分析；（2）采用同位素标记相对和绝对定量(isobaric tags for relative and absolute quantification，iTRAQ) 技术，比较研究了YM-1在适温和低温下的蛋白组，鉴定了204个受低温影响的蛋白，其中包括了跟营养物质转运相关的膜蛋白；（3）成功建立了YM-1的Tn5随机基因插入突变库，但是未鉴定到与低温适应密切相关的蛋白；（4）构建了GFP标记菌株，发现低温刺激有利于耐低温菌在根际的定殖；（5）建立了菌-植互作培养体系，但是未从该体系中检测到群体感应信号物质。取得的主要成果包括：（1）通过组学研究和生理生化试验系统的揭示了耐低温菌YM-1的低温适应机制，包括膜脂不饱和度增加、形成生物膜、合成和吸收更多的分支型氨基酸、增加胞外多糖分泌等;（2）通过体外包涵体表达和重折叠低温菌的外膜蛋白揭示了新的膜蛋白低温适应机制；（3）通过两相培养器培养，蛋白组比较研究和低温刺激栽培实验等证明了菌-藻（植物）互作可缓解藻（植物）的低温胁迫。