关键词: 杨树, 炭疽菌, 遗传转化, 原生质体, 绿色荧光蛋白

Abstract: We established a protoplasts transformation system mediated by polyethylene glycol (PEG), in which poplar anthracnose fungus Colletotrichum gloeosporioides strain C1-5-2 was used as the recipient, and obtained the transgenic transformants expressing a green fluorescence protein (GFP). Protoplasts were mixed with the plasmid gGFP containing the hygromycin phosphotransferase (hph) gene and GFP gene with PEG treatment. Fresh hyphae of recipient strain C1-5-2 were hydrolyzed in 20 mL enzyme solution containing 1% Lysing enzyme for 210 min, by which approximate 10⁸ protoplasts·mL^-1 were obtained. Transformation frequencies of 41 transformants per μg DNA were achieved after being screened on PDA medium containing hygromycin B concentration of 300 μg·mL^-1. The verification with gDNA PCR amplifications indicated that the hph gene and GFP gene were indeed integrated into the genome of the C1-5-2 strain. Fluorescence observation showed clear and strong expression of the green fluorescent protein in fungal structures, and the GFP-tagged transformants were of genetic stability.

Key words: poplar, Colletotrichum, genetic transformation, protoplast, GFP