杨树炭疽病菌原生质体遗传转化的建立及绿色荧光蛋白的表达

摘要

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 l08 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.%以杨树炭疽病菌菌株C 1-5-2作为受体,通过PEG介导的原生质体转化法,将含有潮霉素B(hph)和GFP表达基因的质粒gGFP转入杨树炭疽病菌菌丝的原生质体中.幼嫩菌丝在0.7 mol·L-1 NaC1溶解的1％ Lysing enzyme酶解液的作用下酶解210 min可以得到108· mL-1的原生质体；在PEG介导下,通过含有潮霉素浓度为300μg·mL-1的PDA选择培养基筛选转化子,每微克DNA获得41个转化子的平均转化效率.对转化子进行PCR鉴定表明:hph基因和GFP基因已经整合到杨树炭疽病菌转化子基因组中,通过荧光显微镜观察到转化子可以发出清晰的绿色荧光,且转化子的潮霉素抗性和GFP表达性状可以稳定遗传.