【摘要】 为建立空心莲子草致病菌假隔链格孢菌Nimbya alternantherae的原生质体提取体系,以其强致病性野生型菌株N.alternantherae 11-2为供试菌株,研究了不同菌龄、酶系统、酶解时间及稳渗剂等对假隔链格孢菌原生质体制备及再生的影响,并采用聚乙二醇PEG介导转化法,构建了随机插入突变体库。结果表明,在酵母浸出粉胨葡萄糖液体培养基中培养2 d的假隔链格孢菌丝体,以0.7 M山梨醇为稳渗剂,同时使用2%裂解酶、2%崩溃酶和2%蜗牛酶3种酶的混合液,在30℃下酶解3 h能获得足量的原生质体,且其再生率也可满足后继转化试验的要求。更多还原

【Abstract】 Nimbya alternantherae is a pathogenic fungus of the invasive alligator weed,Alternanthera philoxeroides( Mart.) Griseb. In this study,a highly pathogenic and wild-type strain of N. alternantherae 11-2 was selected to establish a protoplast extraction system. The influences of age,enzyme system, enzymolysis time and osmotic stabilizer on the preparation and regeneration of fungal protoplasts were examined. In addition,the polyethylene glycol-mediated transformation method was used to establish a randomly inserted mutant library. The results showed that sufficient protoplasts could be produced when N. alternantherae hyphostroma were cultivated in a liquid medium of yeast extract peptone dextrose for 2 days and then underwent 3 hours of enzymolysis at 30 ℃,with 0. 7 M sorbierite as the osmotic stabilizer along with a mixture of 2% lyase,2% driselase,and 2% snailase. Furthermore,the regeneration rate of N. alternantherae protoplasts could also meet the requirements of subsequent transformation experiments.更多还原