通过荧光基因标记研究苜蓿中华根瘤菌nifH缺失株的竞争定殖能力
毕业论文关键词：苜蓿中华根瘤菌；nifH；宿主制裁；荧光蛋白标记
Research on the competitive colonization ability of Sinorhizobium meliloti nifH deletion by fluorescent protein labeling
Abstract： Alfalfa is a widly used forage and can form nodules with Sinorhizobium meliloti. If the structure gene nifH of Sinorhizobium meliloti has been deleted, which functions to fix the nitrogen in the air, the symbiotic system including alfalfa and Sinorhizobium meliloti can still form nodules but no nitrogen would be fixed. Thus the Sinorhizobium meliloti is unable to provide its host with enough nitrogen and can be regard as a cheater, which, in microbial ecology, refers to the member making no contribution to its ecosystem. Due to the host sanction theory, the non-nitrogen fixing strains (ΔnifH) will be punished by the host. In this research, Fluorescence protein genes have been successfully marked into the non-nitrogen fixing strains and wild type of Sinorhizobium meliloti, which was mCherry and gfp respectively. Both these two strains has been inoculated into the alfalfa and their living conditions in the nodules has been observed, the result shows that the fluorescent protein genes can be successfully expressed, which may pave the way for further discussion to the host sanction theory.
Key words: Sinorhizobium meliloti；competitive colonization；nifH；Fluorescent protein labeling
目 录
摘要3
关键词3
Abstract3
Key words3
引言（或绪论）3
1□材料与方法5
1.1□供试菌株 5
1.2□培养基和培养条件5
1.3□苜蓿所用培养液及培养条件5
1.4□抗生素与浓度6
1.5□荧光蛋白基因的标记6
1.5.1□质粒DNA少量提取6
1.5.2□根瘤菌感受态细胞的制备和电刺激转化6
1.5.3□蓝白斑筛选法6
1.5.4□荧光蛋白基因的插入根瘤菌基因组6
1.5.5□根瘤菌与大肠杆菌的双亲接合7
1.6□标记菌株与出发菌株生长曲线的比较7
1.7□苜蓿的培养与根瘤菌的接种7
1.7.1□紫花苜蓿种子催芽7
1.7.2□紫花苜蓿的培养 7
1.8□激光共聚焦扫描显微镜观察标记菌定殖情况7
2□结果分析7
2.1□构建荧光蛋白标记根瘤菌菌株 8
2.1.1□构建△nifH-mCherry荧光蛋白标记菌株8
2.1.2□构建2011-GFP荧光蛋白标记菌株10
2.2□荧光蛋白标记菌株生长曲线 11
2.3□荧光蛋白标记的根瘤菌在根瘤中的定殖情况12
3□讨论13
致谢13
参考文献13
通过荧光基因标记研究苜蓿中华根瘤菌nifH缺失株的竞争定殖能力
引言:自然界中不同物种之间的互利共生关系是普遍存在的，其中豆科植物和根瘤菌的共生体系因为其结瘤固氮的特性一直受到关注。苜蓿是豆科苜蓿属（Medicago）植物的通称，多年生开花植物，是良好的牧草材料和绿肥，可以与苜蓿中华根瘤菌共生形成根瘤。[1]
通常，与互利细菌组成的共生体系可以显著地影响动植物宿主的相关表型[2]，也就是说，当宿主与互利细菌形成共生体后，他们就可以合作以更好地共享资源。但是有时共生体系中会存在寄生关系，即寄生菌种不能够有效地向宿主提供足够的营养物质，这种寄生菌株可被认为是欺骗株（Cheater）[3]，例如在豆科植物-根瘤菌共生体系中，寄生的根瘤菌无法向豆科宿主提供氮素，破坏了正常的碳氮交互关系；而且固氮共生体消耗宿主的碳源资源极高，所以宿主必然会对共生体系中的寄生行为进行制裁，即宿主制裁。[4]