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【目的】摸索葡萄溃疡病菌(Lasiodiplodia theobromae)限制性内切酶介导整合(Restriction enzyme mediated integration,REMI)的转化方法,并构建CSS-01s(L.theobro-mae)的REMI转化子库。【方法】利用REMI转化方法,将线性化的含有潮霉素抗性基因(Hygromycin phosphotransferase gene,Hyg)的pUCATPH质粒转化CSS-01s菌株的原生质体;测定其对潮霉素B的敏感浓度,摸索不同酶解液和酶解时间对原生质体制备的影响,统计不同限制性内切酶酶量对转化效率的影响,以摸索出的最优条件构建CSS-01s菌株的REMI转化子库,采用Southern blot的方法验证转化子。【结果】首次建立了一套L.theobromae的REMI转化方法,经转化得到包含6 000余个转化子的L.theobromae CSS-01s转化子库,随机挑选的5个转化子经Southern blot分析,质粒均插入到了HindⅢ相应的酶切位点处。【结论】浓度为2%崩溃酶+2%蜗牛酶酶混合液、酶解4 h为原生质体获得的最优条件,每管转化体系中加入30 U HindⅢ时转化效率最高,该方法可以用来获得大量不同表型的REMI转化子。
【Objective】 To explore the transformation method of Restriction enzyme mediated integration (REMI) in Lasiodiplodia theobromae and to construct REMI transformant library of CSS-01s (L.theobro-mae). 【Method】 The linearized pUCATPH plasmid containing hygromycin phosphotransferase gene (Hyg) was transformed into protoplasts of CSS-01s strain by REMI transformation method. The sensitive concentration of hygromycin phosphotransferase gene (Hyg) Effects of different enzymatic hydrolysis and enzymolysis time on the preparation of protoplasts were investigated. The effects of different restriction enzyme amount on the transformation efficiency were calculated. The REMI transformant library of CSS-01s strain was constructed based on the optimized conditions. Transformants were verified by the method of blot. 【Result】 A set of REMI transformation method of L. theobromae was established for the first time. After transformation, the library of L. theobromae CSS-01s containing more than 6000 transformants was obtained. Five randomly selected transformants were analyzed by Southern blot. Plasmids Were inserted into the Hind Ⅲ corresponding restriction sites. 【Conclusion】 The optimal conditions for protoplast hydrolysis were obtained with 2% catalase and 2% snail enzyme mixture, and the optimal transformation conditions were obtained after 4 h of enzymatic hydrolysis. When 30 U HindIII was added into each transformation system, the transformation efficiency was the highest, A large number of REMI transformants of different phenotypes were obtained.