青枯雷尔氏菌(Ralstonia solanacearum) 绿色荧光蛋白标记研究教材.doc

青枯雷尔氏菌（Ralstonia solanacearum） 绿色荧光蛋白标记研究 （福建省农业科学院，福州 350003） GFP Tagging Ralstonia solanacearum with gfp/luxAB mini-Tn5 CHE Jian-mei1,2, LAN Jiang-lin1, LIU Bo1 (1Agricultural Bioresource Research Institute, Fujian Academy of Agricultural Sciences, Fuzhou 350003; 2Key Laboratory of Biopesticide and Chemical Biology, Fujian Agricultrue and Forestry University, Ministry of Education, Fuzhou 350002) Abstract: 【Objective】The Ralstonia solanacearum strain was chromosomally tagged with the marker genes gfp and luxAB by electroporation. The growth properties and the pathogenicity of the tagged strain and the wild type were compared in this paper.【Method】The R solanacearum strain was transformed by the electroporation. The condition and processor of the invasion of the GFP-tagged strain was observed in the tomato tissue culture plants. 【Result】 Transformant was screened for the green fluorescence by fluorescence stereomicroscopy. A 3.0 b fragment of the gfp gene and luxAB gene was amplified from the genome DNA of the GFP-tagged strains. Morphologically, the cells of the engineered strain were similar to wild type strain as determined by laser scanning confocal microscopy. Luciferase activity of the GFP-tagged strain increased rapidly during the log phase, but decreased in the stationary phase. The green fluorescence could be kept until 20 times of subcultures. It was the same for the GFP-tagged strian and the wild type strain in the culture temperature, pH value and growth curve. These results confirmed that the dual marker was inserted successfully into the R. solanacearum strain. The pathogenicity of the wild type strain and the GFP-tagged strain was over 90% which means gfp gene had no effect on the pathogenicity. 【Conclusion】The GFP-tagged R solanacearumis obtained in this study. The growth propertities and the pathogenicity of the GFP-tagged strain is the same to the wild type strain. Key words: Ralstonia solanacearum; Electroporation; gfp/luxAB ge