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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。
In order to establish the protoplast extraction system of Alternaria solani of Alternanthera philoxeroides, its virulent wild-type strain N.alternantherae 11-2 was used as the tested strain, , Enzymolysis time and stabilizing agent on the preparation and regeneration of protoplast of Alternaria alternata, and the random insert mutant library was constructed by polyethylene glycol PEG-mediated transformation. The results showed that in mycelium of yeast extract peptone dextrose and glucose culture medium for 2 days, Mycelial septoria tritici were cultured with 0.7 M sorbitol as stabilizing agent, 2% lyase, 2% catabolism enzyme and 2% Snail enzyme mixture of three kinds of enzymes, enzymatic hydrolysis at 30 ℃ 3 h get enough protoplasts, and its regeneration rate can also meet the requirements of subsequent transformation test.