GFP标记生防细菌B579及其定殖能力检测

 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。

GFP-expressing Bacillus subtilis B579 strain and its colonization detection

GFP (Green fluorescent protein) is described as one of the important methods to study the interaction between target microbe and host. The shuttle vector pGFP4412, carrying gfpmut3a gene, was transformed into Bacillus subtilis B579 strain and expressed successfully. B579-gfp colonization on the surface of cucumber roots was conducted by combining antibiotics plate recovery and Laser Scanning Confocal Microscope (LSCM). The results showed that GFP-expressing B579-gfp was observed under 488 nm wavelength on the surface of cucumber roots, root bifurcated place and the pileorhiza. The B579-gfp could be recovered by antibiotics plate from three different inoculation ways (seeds soaking dipping roots and pouring roots), the numbers recovered were 4.0?10³ , 1.0?10⁴ and 2.0?10² cfu/g, respectively.