含有增强型绿色荧光蛋白报告子的载体系统的构建

Construction of a plasmid system containing reporter of enhanced green fluorescent protein

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 enhanced green fluorescent protein （EGFP） was cloned into the promoterless vector pLAFR6, generated the reporter plasmid pLZY. The promoter, secretion and translocation signal of xopXccN, a known type Ⅲ effector in Xanthomonas campestris, were cloned into pLZY. The recombinant plasmid was transferred to Xanthomonas campestris and inoculated in radish. Green fluorescence was observed in the positive colonies under fluorescent microscopy and in the plant cell under confocal laser scanning microscope. It confirmed that the reporter plasmid pLZY work well. This reporter plasmid provides useful vessel to study the expression of target genes in different hosts and can be used to study the subcellular localization of the type Ⅲ effector proteins of X. campestris in plants.