利用细菌噬菌体ΦRSS1来源的绿色荧光蛋白质粒标记植物病原性青枯雷尔氏.doc

利用细菌噬菌体ΦRSS1来源的绿色荧光蛋白质粒监测植物病原菌青枯雷尔氏菌细胞 Takeru Kawasaki1, Hideki Satsuma1, Makoto Fujie1, Shoji Usami1, and Takashi Yamada1* Department of Molecular Biotechnology, Graduat School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8530, Japan1 本研究从可以侵染植物病原菌青枯雷尔氏菌的丝状细菌噬菌体ΦRSS1中构建了绿色荧光蛋白（green fluorescent protein, GFP）表达质粒。这个质粒为pRSS12（大约4.7 kbp），由2248 bp的ΦRSS1 RF DNA组成，包括ORF1-ORF3和插入序列（intergenic region，IG），以及Km区域和GFP基因。本文通过电击方法将质粒导入青枯雷尔氏菌，发现这个质粒可以在无选择压力的条件下在不同的生理小种和不同生物群的青枯雷尔氏菌中稳定表达。可以从侵染的番茄组织（包括茎部、叶柄和根部）以及土壤中检测到强烈的绿色荧光。这些结果表明，不管在细胞学研究还是在田间研究，pRSS12均可作为任何给定的青枯雷尔氏菌的易操作的GFP标记工具。 [关键词：细菌监测，绿色荧光蛋白，噬菌体来源质粒，青枯病，青枯雷尔氏菌 Monitoring of Phytopathogenic Ralsonia solanacearum Cells Using Green Fluorescent Protein-Expressing Plasmid Derived from Bacteriophage ΦRSS1 Takeru Kawasaki1, Hideki Satsuma1, Makoto Fujie1, Shoji Usami1, and Takashi Yamada1* Department of Molecular Biotechnology, Graduat School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8530, Japan1 A green fluorescent protein (GFP)-expressing plasmid was constructed from a filamentous bacteriophage ΦRSS1 that infects the phytopathogen Ralstonia solanacearum. This plasmid designated as pRSS12 (4.7 kbp in size) consists of an approximately 2248 bp region of the ΦRSS1 RF DNA, including ORF1-ORF3 and the intergenic region (IG), and a Km cassette in addition to the GFP gene. It was easily introduced by electroporation and stably maintained even without selective pressure in strains of R. solanacearum of different races and biovars. Strong green fluorescence emitted from pRSS12-transformed bacterial cells was easily monitored in tomato tissues (stem, petiole, and root) after infection as well as from soil samples. Theses results suggest that pRSS12 can serve as an easy-to-use GFP-tagging tool for any given strain of R. solanacearum in cytological as well as field studies. [Key words: bacterial monitoring, green fluorescent protein (G