葡萄溃疡病菌限制性内切酶介导整合方法的建立及转化子库的构建

Establishment of restriction enzyme mediated integration and construction of transformants library in Lasiodiplodia theobromae

【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 establish the Restriction enzyme mediated integration （REMI） trans- formation system and a REMI transformants library of Lasiodiplodia theobromae. [Methods] REMI method was used to obtain transformants of L. theobromae. Linearized DNA of plasmid pUCATPH containing the hygromycin phosphotransferase gene （Hyg） was integrated into chro- mosomes of wild strain CSS-01s. Sensitive concentration of L. theobromae to hygromycin B was determined. Different enzymolysis systems and digest time on preparation of protoplasts were performed and effect of restriction enzyme quantity on percent conversion was also carried out. The REMI transformants library of CSS-01 s was constructed with the optimal condition, and the transformants was confirmed by Southern blot. [Results] This study established REMI method of L. theobromae for the first time and constructed REMI transformants library containing about 6 000 transformants. Southern blot analysis revealed that the plasmid inserted into the corresponding restriction endonuclease sites located on the genomic DNA of five random transformants. [Con- clusion] The results suggested that the optimal condition ofprotoplasts preparation was adding 2% Drislase＋2% Snailase enzyme mixture and digested for 4 hours. The transformation efficiency reached to the highest when 30 U Hind Ⅲ was added into per transformation reaction. Transfot- mants with differernt phenotype can be obtained using the above optimal conditions.