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将增强型绿色荧光蛋白基因编码序列克隆到不含启动子的载体pLAFR6上,构建报告质粒pLZY。将野油菜黄单胞菌的已知效应物基因xopXccN启动子和分泌转运信号区序列克隆到pLZY,重组质粒导入野油菜黄单胞菌,阳性克隆细胞在荧光显微镜下呈现绿色荧光。阳性克隆接种萝卜叶片,在共聚焦激光扫描显微镜下检测观察到在植物细胞内发荧光,而含有pLZY的野油菜黄单胞菌对照菌株菌体和植物细胞内都检测不到绿色荧光,证明报告质粒pLZY工作正常。该报告质粒为研究目的基因在不同宿主中的表达和研究植物病原细菌Ⅲ型效应物蛋白的植物亚细胞定位提供了材料。
The coding sequence of the enhanced green fluorescent protein gene was cloned into the promoter-free vector pLAFR6 to construct the reporter plasmid pLZY. Xanthomonas campestris known effector gene xopXccN promoter and secretory transport signal sequence was cloned into pLZY, the recombinant plasmid was introduced into Xanthomonas campestris, the positive clonal cells under the fluorescence microscope showed green fluorescence. Positive clones were inoculated with radish leaves and fluorescence was observed in the plant cells under confocal laser scanning microscopy whereas green fluorescence was not detected in the cells and plant cells of X. campestris pLZY containing pLZY, Plasmid pLZY is working properly. The reporter plasmid provided the material for studying the expression of the gene of interest in different hosts and for studying plant subcellular localization of plant pathogenic bacteria type III effector proteins.