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利用绿色荧光蛋白(Green fluorescent protein,GFP)标记靶标微生物是目前研究微生物和宿主互作的重要手段。在本研究中,作者用电击转化的方法将携带质粒gfpmut3a基因的穿梭载体pGFP4412导入生防枯草芽孢杆菌(Bacillus subtilis)B579中,并得到成功表达GFP的枯草芽孢杆菌B579-gfp。用抗生素平板回收结合激光扫描共聚焦显微镜(LSCM)观察对枯草芽孢杆菌B579-gfp在盆栽的黄瓜根表定殖情况进行了研究,结果表明:标记菌株在激发光波长为488 nm的蓝光下可观察到亮绿色的荧光;B579-gfp菌体能够定殖在黄瓜根部,在根基部和中部都有菌体聚集而形成膜状结构,在根的分叉和根冠处可观察到大量B579-gfp定殖;浸种、蘸根以及灌根接种均可回收到大量的B579-gfp,分别为4.0×103、1.0×104和2.0×102 cfu/g。
The use of green fluorescent protein (GFP) to label target microorganisms is an important method to study the interaction between microorganisms and host. In the present study, the authors introduced the shuttle vector pGFP4412 carrying the gfpmut3a gene into Bacillus subtilis B579 by electroporation and obtained Bacillus subtilis B579-gfp successfully expressing GFP. The antibacterial plate recovery combined with laser scanning confocal microscopy (LSCM) observed Bacillus subtilis B579-gfp colonization of potted cucumber root table results showed that: the marker strain in the excitation wavelength of 488 nm blue light The bright green fluorescence was observed. The B579-gfp cells colonized in the cucumber root, and the bacterial cells aggregated and formed membranous structures in the root and the middle. A large number of B579- gfp colonization. B579-gfp was recovered in soaking seeds, dipping roots and irrigating roots, respectively, which were 4.0 × 103, 1.0 × 104 and 2.0 × 102 cfu / g, respectively.