为了探索不同酶系组成对玉米弯孢叶斑病菌原生质体制备的影响,建立该病菌原生质体遗传转化系统,采用酶系混合物裂解菌丝体制备原生质体,采用PEG介导方法进行原生质遗传转化,通过PCR和Southern blotting技术对转化子进行验证.结果表明:1％溶壁酶+1％蜗牛酶+1％纤维素酶混合酶系为玉米弯孢叶斑病菌原生质体制备的最佳酶系,可以产生原生质体6.78×106 du/mL.用PEG介导方法转化共获得16个稳定的转化子,从中随机挑取5个转化子发现质粒pV2已被成功整合到基因组中.本研究获得了制备玉米弯孢叶斑病菌原生质体的最佳酶系,建立了PEG介导的原生质体遗传转化体系,为开展该菌致病相关基因克隆和基因功能研究提供了一种手段.%The effects of the composition of various enzymes on protoplast of Curvularia lunata preparation were explored, and its protoplast-mediated genetic transformation system was established. Protoplast was obtained through cleavage of mycelium using enzyme mixture. Protoplast genetic transformation was mediated by polyethylene glycol (PEG), and transformants were analyzed by PCR and Southern blotting. The best composition of enzymes for protoplast preparation was 1% snailase, 1% cellulase and 1% wallzyme, which could produce 6.78× 106 protoplasts/mL. Sixteen stable transformants were obtained by PEG, and the plasmid pV2 was successfully integrated into the genome in 5 randomly selected transformants. The best composition of enzymes for protoplasts preparation was obtained, and the protoplast-mediated genetic transformation system of C. lunata was established, which provided a tool for cloning pathogenicity genes and studying the gene function.
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