水稻内生优势成团泛菌GFP标记菌株的性质与标记丢失动力学

Characteristics and Label Loss Dynamics of Pantoea agglomerans, Predominant Endophytic Bacteria Isolated from Rice Plant

为研究内生细菌对宿主植物侵染定殖的机理和其共生生物学作用 ,对水稻内生优势成团泛菌 (Pantoeaagglomerans)YS19与绿色荧光蛋白 (GFP)标记的YS19B ::gfp菌株的生长动力学进行了比较研究 ,探讨了成团泛菌YS19B ::gfp的标记稳定性和荧光性质 .标记菌株与野生型菌株相比 ,最大比生长速率和最大生物量仅减小 12 4 %和 6 % ,代时延长 14 0 % .成团泛菌YS19B ::gfp在指数期连续传代培养 10 0代后 ,GFP标记的保持率为 89 1% ,建立了标记菌株在有标记丢失存在时的生长动力学模型 :dX+ dt =μ+ (1-p)X+ ,解析出细胞分裂时标记丢失的概率p =9 75 6× 10 -7,确定了方程的模型参数 .标记菌株的荧光光谱在激发波长为 4 0 0nm时 ,最大发射波长为 5 0 8nm ,与供体菌株完全相同 .在LB培养基上生长时 ,成团泛菌YS19B ::gfp的GFP产生时间在指数期末期到稳定期较快 ,并于培养至 2 0h时达到最高 ,同时单位菌体生物量的荧光强度也达到最大 .结果说明 ,在GFP标记后成团泛菌YS19B ::gfp的生长仅受到较小影响 ,不致对成团泛菌的生理活动造成大的改变 ,同时由于该菌对宿主的侵染能力比其它内生细菌要强得多 ,因而该菌对植物的侵染活性影响也较小 ,该菌仍然可以保持其内生优势地位 .该标记的稳定性比较高 ,荧光产生正常 ,很适?

In order to reveal the infection and living mechanism and the biological effects of endophytic bacteria, the key point was to provide a stable labelled model endophytic bacterium. Pantoea agglomerans bacteria tagged with gfp were studied, whose wild type (wt) bacteria were predominant bacteria isolated from their host rice ( Oryza sativa ). The growth dynamics, the stability of the green fluorescent protein(GFP) label and the fluorescence were studied. The results showed that the maximal growth rate and biomass in LB medium reduced 12 4% and 6% in comparison with the wild type strain, meanwhile, the generation time was about 14 0% longer than that of the wild type strain. About 89 1% GFP label was constant in P. agglomerans YS19B:: gfp when they were continuously transferred in LB medium and proliferated for 100 times. A dynamics equation model describing the growth of YS19B:: gfp accompanying the loss of gfp , was established as follows: d X +/d t = μ + (1 - p)X + . The model parameter p , indicating the loss rate of the gfp was 9 756×10 -7 . The fluorescence spectra of P. agglomerans YS19B:: gfp showed that the maximal emission wavelength was 508 nm when excited at 400 nm, which was the same as the gfp donator strain. GFP was produced quickly by P. agglomerans YS19B:: gfp during the time from the end of the exponential growth phase to the stationary growth phase and reached the maximal value after being cultured for 20 hours in LB medium, when fluorescence per cell dry weight also reached the highest value. These results showed that the growth rate was affected so slightly that the predominance of these bacteria in the endophytic bacteria of plant rice could be conserved because of its high infection and colonization ability. With rather high constancy of labelled gene and proper fluorescence producing, YS19B:: gfp was suitable to visualize its interaction with the host plant.