微生物学通报运用萎锈灵抗性基因构建香菇聚乙二醇介导.pdf

微生物学通报 Aug. 20, 2018, 45(8): 1824−1828 Microbiology China /wswxtbcn tongbao@ DOI: 10.13344/j.microbiol.china.170861 生物实验室 运用萎锈灵抗性基因构建香菇聚乙二醇介导的遗传转化方法 鲍大鹏 杨瑞恒 王莹 茅文俊 汪滢 李燕* ( 201403) 摘 要：【背景】萎锈灵抗性基因作为筛选标记在植物和真菌中得到广泛应用。【目的】构建 可以使用萎锈灵作为筛选标记的香菇遗传转化技术。【方法】利用溶壁酶消化培养4 d 的香菇 菌株411-4 的菌丝体获得原生质体，加入适量pL-cbx 质粒和聚乙二醇溶液，混合物涂布于再 生筛选培养基上，培养后挑选菌落进行验证试验。【结果】在原生质体数目108 个和添加4 µg 质粒DNA 的情况下，得到了40 个抗性转化子。利用PCR 实验和转代实验对转化子进行验证， 结果显示38 个转化子的抗性可稳定遗传，表明萎锈灵抗性基因整合进入了供试菌株的基因组 中。【结论】利用香菇411-4 菌株建立了一套运用萎锈灵抗性基因作为分子标记的遗传转化技 术体系。 关键词：香菇，聚乙二醇介导的原生质体转化，萎锈灵 PEG-mediated genetic transformation of Lentinula edodes by using carboxin resistant gene as selective marker * BAO Da-Peng YANG Rui-Heng WANG Ying MAO Wen-Jun WANG Ying LI Yan (National Engineering Research Center of Edible Fungi, Key Laboratory of Edible Fungi Resources and Utilization (South), Ministry of Agriculture, Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, Shanghai 201403, China) Abstract: [Background] As a selective marker, carboxin has been used in plant and fungi transformation. [Objective] To construct the genetic transformation system of Lentinula edodes by using carboxin resistant gene as a selective marker. [Methods] The mycelia of L . edodes strain 411-4 th were collected at the 4 day after cultivation and then digested by lywallzyme to obtain the protoplast. Sufficient protoplast was mixed with the plasmid pL-cbx DNA and the