GFPuv标记猕猴桃溃疡病菌的生物学特性 及其在土壤、根系中的定殖

【目的】获得具有荧光标记的猕猴桃溃疡病菌（Pseudomonas syringae pv. actinidiae）菌株，为进一步揭示其侵染致病过程奠定基础。【方法】电击法对病菌进行GFPuv基因标记，应用荧光显微镜和平板稀释法研究标记菌株在土壤和根部的定殖情况。【结果】GFPuv标记的猕猴桃溃疡病菌菌株在荧光显微镜下发出强烈的绿色荧光，8株标记菌基因组DNA中均扩增出约700 bp的目的片段。标记菌株PSAmx7-GFPuv1的菌体形态、生长曲线、最适温度、最适pH、致病性均与野生型无显著差异，其绿色荧光可稳定遗传。标记菌在灭菌土壤中可存活3个月左右，在未灭菌土壤中也能存活3周；灌根1 d检测，根表、根内组织中可分离到目标菌落，随后标记菌株数量呈现“先增后降”的趋势。【结论】电击法成功地将GFPuv基因转入猕猴桃溃疡病菌；导入的GFPuv 基因对宿主菌的生物学特性没有影响；标记菌可在灭菌土壤中长期存活，并在根部定殖和增殖。

Transformed GFPuv into Pseudomonas syringae pv. actinidiae and Its Biological Characteristics and Colonization in Soil and Roots of Kiwifruit

【Objective】The objective of this study is to obtain fluorescence-labeled Pseudomonas syringae pv. actinidiae (Psa) strain to further understand the infection and pathogenic processes.【Method】The Psa strain was tagged with GFPuv by electroporation. Fluorescence microscope and the method of plate dilution were used to detect the colonization of tagged strain in soil and root. 【Result】 The transformants showed strong green fluorescence under UV-light indicating GFPuv expression. A 700 bp fragment of the GFPuv gene was amplified from the genome DNA of the transformants. There were no differences between the engineered PSAmx7-GFPuv1 strain and wild-type strain including morphology, optimum temperature, pH and pathogenicity. The green fluorescence could be kept until 20 times of subcultures. The PSAmx7-GFPuv1 could survive in sterile soil for three months, and three weeks or so in non-sterile soil. The PSAmx7-GFPuv1 also could infect and colonise in root.【Conclusion】The GFPuv-tagged Psa was obtained successfully through electroporation in this study. The presence of GFPuv did not have signi?cant impact on the wild-type strain. The tagged strain could survive for a long period of time in sterile soil, colonise and multiply in root.