中国生物防治学报Issue(2)：263-269,7.
限制性内切酶介导的粉红粘帚霉67-1转化体系构建
Construction of Clonostachys rosea 67-1 Genetic Transformation System by Restriction Enzyme-mediated Integration (REMI)
摘要
Abstract
Authors tried to genetically transform mycoparasitic fungus Clonostachys rosea 67-1 with hygromycin B resistance gene hph vectored in plasmid pAN7-1 via restriction enzyme-mediated integration. Results showed that content of polyethylene glycol (PEG), type and concentration of restriction endonuclease, plasmid structure, and reaction time showed significant effects on the transformation. When linear pAN7-1, 40% PEG3350 solution, 20 U HindⅢ and 10 min for reaction were adopted, efficiency of the transformation was 30—40 CFU·μg−1 plasmid DNA. Continuous subcultures for 3 times on PDA plates, followed by culturing on hygromycin resistance plates indicated that the transformants obtained were genetically stable. PCR, Southern blotting, and Real-time PCR analyses showed that hph gene was successfully integrated into the genomic DNA of the transformants,in most cases by single copy insertion. Observation on fifty randomly selected transformants on PDA plates demonstrated that 20% transformants grew faster and 12% sporulated more in comparison with their parent strains. This may provide an effective protocol for integration of exogenous genes into C. rosea 67-1 and related fungal species.关键词
粉红粘帚霉/原生质体转化/限制性内切酶介导整合/潮霉素抗性Key words
Clonostachys rosea/genetic transformation/restriction enzyme-mediated integration/hygromycin B resistance分类
农业科技引用本文复制引用
许梦秋,姜杰,孙漫红,谢响明,李世东..限制性内切酶介导的粉红粘帚霉67-1转化体系构建[J].中国生物防治学报,2013,(2):263-269,7.基金项目
转基因重大专项（2009ZX08009-0898）；国家“863”计划项目 （）
