利用GFP表达系统检测球孢白僵菌侵染昆虫过程

GFP as a vital marker for investigation of pathogenic development of Beauveria bassiana

绿色荧光蛋白(GFP)作为一种重要的报告基因,在病原菌和寄主之间的互作研究中具有良好的应用前景。利用草丁膦抗性基因bar为筛选标记,构建了组成性表达绿色荧光蛋白基因egfp的载体pBG,并转入球孢白僵菌获得成功表达。将转化子接种菜青虫进行生物测定表明,组成性表达egfp不影响球孢白僵菌的毒力。进一步利用冰冻切片和荧光显微观察技术监测球孢白僵菌侵染寄主行为,结果显示,通过荧光观察可清楚的检测到病原菌在昆虫体表的附着、菌丝穿透体壁以及菌丝从寄主体内长出等侵染过程。由此表明,GFP可有效用于球孢白僵菌的遗传标记,进行病原菌的鉴别、侵染致病机理及相关功能基因的表达模式研究。

The green fluorescent protein （GFP） has been expressed in a variety of organisms and has been used extensively as a marker in the study of host-pathogen interaction. We have expressed GFP in the entomopathogenic fungus Beauveria bassiana through transformation with a vector that confers resistance to phosphinothricin. Conidia expressed GFP and were readily detected by fluorescence microscopy. Fluorescent hyphae and conidia were easily distinguished in the tissue of the cabbage worm. GFP-tagged strains of B. bassiana could be used to study the developmental fate of the fungus within its insect hosts. Thus, this method is useful for screening and monitoring the engineered strains even after infection.