丛枝菌根真菌状巨孢囊霉孢子伴生细菌的绿色荧光蛋白标记及定殖分析

Analysis of bacterial colonization associated with Gigaspora margarita spores by green fluorescence protein (GFP) marked technology

摘要:

摘要：【目的】分析丛枝菌根（Arbuscualr Mycorrhizal, AM）真菌珠状巨孢囊霉(Gigaspora margarita) MAFF 520054孢子伴生细菌的定殖情况，明确其生态位点，以及为进一步分析其种群生态或功能提供信息。【方法】以载体pNF8（gfp-mut1）对6株珠状巨孢囊霉MAFF 520054 孢子伴生细菌进行绿色荧光蛋白（GFP）标记，并通过荧光显微镜和平板计数的方法研究标记菌株对真菌宿主的定殖位点和不同条件下的定殖数量动态。【结果】对粘状芽孢杆菌（Peanibacillus spp.）M060106-1和M061122-6、芽孢杆菌（Bacillus sp.）M061122-10和短小芽孢杆菌（Brevibacillus sp.）M061122-12成功进行了GFP标记，其均具有较好的质粒稳定性，且与出发株的基本性状一致，适合短期内进行环境定殖研究。所有菌株均能定殖珠状巨孢囊霉MAFF 520054孢子壁，而M061122-6和M061122-12还能够定殖其菌丝；不同pH值条件下，各菌株定殖珠状巨孢囊霉MAFF 520054孢子的数量动态均为先上升后下降，pH值对各菌株的定殖数量有不同的影响；各GFP菌株对低活力的珠状巨孢囊霉孢子定殖数量高于高活力的孢子，且对高活力孢子的定殖数量动态不同。【结论】分离的珠状巨孢囊霉孢子伴生细菌能够重新定殖其孢子，菌株的定殖能力受其特性及外界因子的影响，为进一步分析AM真菌伴生细菌的种群生态及功能提供了信息。

Abstract:

Abstract: [Objective] We analyzed bacterial colonization associated with spores of arbuscular mycorrhizal fungi (AMF) Gigaspora margarita, to indicate their ecological niche, and to provide information for further researches on their populations or functions. [Methods] Six bacteria strains (Peanibacillus sp. M060106-1, Peanibacillus sp. M061122-2, Peanibacillus sp. M061122-6, Bacillus sp. M061122-4, Bacillus sp. M061122-10 and Brevibacillus sp. M061122-12) isolated from G. margarita spores were tagged with green fluorescence protein (GFP) using the carrier plasmid pNF8 (gfp-mut1). We analyzed the ecological niche and population dynamics of tagged strains on G. margarita under different conditions by using fluorescent microscope and/or plate counts. [Results] Four strains (M060106-1, M061122-6, M061122-10 and M061122-12) were tagged with GFP, showing high plasmid stability. These tagged strains possessed the basic characteristics identical to their original strains and, hence, were fit for short-term study of environmental colonization. All four GFP-tagged strains colonized the spore wall of G. margarita, and M061122-6 and M061122-12 further colonized the fungal hyphae. Under different pH conditions, the population dynamic of each GFP-tagged strain on the spores showed the same trend, i.e. first increased and then decreased, and the effects on the population size varied with different pH value. GFP-tagged strains colonized the spores of low viability more easily than those of high viability, and the population dynamic on the spores of high viability was different for each tagged strain. [Conclusion] The isolated bacteria associated with G. margarita spores can re-colonize the fungal spores, whereas their colonizing ability depends on their characteristics and environmental factors. These data contributes to the further understanding of populations and functions of AMF-associated bacteria.