芒果尖孢炭疽病病原菌的绿色荧光蛋白基因标记

Green Fluorescent Protein Transformed Colletotrichum acutatum from Mango

芒果炭疽病是世界性的主要病害之一,引起芒果炭疽病的病原菌有胶孢炭疽菌和尖孢炭疽菌2种。通过溶壁酶酶解菌丝体成功制备尖孢炭疽菌原生质体,用抗潮霉素基因作为选择标记,采用PEG介导的原生质体转化法,用绿色荧光蛋白表达载体(pCT74-sGFP)转化原生质体,获得表达GFP尖孢炭疽菌的转化子菌株;转化子菌株在含潮霉素平板上经多次单孢纯化后,在无选择压下连续继代培养仍能发出稳定而强烈的绿色荧光,用GFP特异性引物PCR扩增转化子菌株基因组DNA获得预期大小片段,表明gfp基因已成功导入芒果炭疽病病原菌基因组中,且稳定遗传;GFP标记菌株生长正常,致病性和野生型菌株无明显差别,这为进一步研究该病菌在自然界的生物学及侵染方式奠定了基础。

Mango anthracnose is a major disease on mango （Mangifera indica L.）, caused by Colletotrichum gloeosporioides and（or）C, acutatum. The protoplasts of a C. acutatum the strain T7 were prepared by digesting the mycelia with lywallzyme. Furthermore, the expression vector pPCT74-sGFP was transformed into the protoplasts with PEG mediated method. Transformation were regenerated and selected on hygromycin-containing medium. Single-spore isolates obtained and showed strong green fluorescence after several successive generations on the medium without hygromycin. The results showed that pPCT74-sGFP was successfully integrated into the strain T7. The presence of the green fluorescent protein DNA in the fungal cells was confirmed by PCR using a GFPspecific primer pair. The transformed isolates did not differ markedly from the wild type isolate in growth and morphological characteristics in vitro. Pathogenicity tests showed that the transformation process did not alter virulence of the strain T7.