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

PEG-mediated genetic transformation of Lentinula edodes by using carboxin resistant gene as selective marker

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。

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 were collected at the 4th day after cultivation and then digested by lywallzyme to obtain the protoplast. Sufficient protoplast was mixed with the plasmid pL-cbx DNA and the polyethylene glycol (PEG) solution for transformation. The mixture finally was spread on the regeneration selecting plate, and the generated colonies were picked up for further testing. [Results] When more than 108 protoplasts and 4 μg plasmid DNA were used, 40 transformants were obtained. After being subcultured on media under carboxin selective stress, and the PCR identification, 38 transformants were positive and stable, indicating that carboxin gene was integrated into the genome of L. edodes 411-4. [Conclusion] The PEG mediated transformation method was constructed by using L. edodes strain 411-4 and carboxin selective marker.