猕猴桃间座壳菌Diaporthe actinidiae的绿色荧光蛋白基因标记及侵染过程

果实软腐病是猕猴桃贮藏期间最严重的真菌病害,猕猴桃间座壳Diaporthe actinidiae是该病检出率最高且致病力最强的病原菌.该病菌从花前期开始侵染,至果实贮藏期才表现症状,侵染至发病周期较长,可借助荧光标记对其侵染过程进行研究.本研究采用PEG介导原生质体转化的技术,运用携带GFP及潮霉素抗性基因的双元载体p...

Green fluorescent protein gene markers and infection of Diaporthe actinidiae

Fruit soft rot is the most serious fungal disease of kiwifruit during storage period. Diaporthe actinidiae is the pathogen with the highest detection rate and the strongest pathogenicity. The pathogen invaded before florescence and showed symptoms until fruit storage. The long infection process can be studied with the help of fluorescent markers. In this study, PEG mediated protoplast transformation of D. actinidiae strain was performed by applying recombinant vector pCAMBgfp carrying GFP and hygromycin gene. The transformants were screened by hygromycin and GFP fluorescence, and then the colony morphology, stability of fluorescence and pathogenicity were analyzed. Three positive transformants showing no significant difference from wild strain in colony morphology, growth rate and pathogenicity were screened out. The transformant KFDA-17 was inoculated to kiwifruit ‘Jinyan’ during storage period. After inoculation, the fruit appeared watery symptoms in 1 d, and soft rot appeared and gradually accelerated. Mycelium showed obviously green fluorescent under fluorescence microscope, and the infection process can be clearly observed. The result proved that the PEG-mediated protoplast transformation of D. actinidiae was successfully constructed, and the obtained fluorescent strains KFDA-17 could be used for the study of infection process.