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)

作　　者：许梦秋[1,2] 姜杰[1,2] 孙漫红[1] 谢响明[2] 李世东[1] 

机构地区：[1]中国农业科学院植物保护研究所/农业部作物有害生物综合治理重点实验室,北京100081 [2]北京林业大学生命科学学院,北京100083

出　　处：《中国生物防治学报》2013年第2期263-269,共7页Chinese Journal of Biological Control

基　　金：转基因重大专项(2009ZX08009-0898);国家"863"计划项目(2011AA10A205)

摘　　要：本文将含有潮霉素抗性基因(hph)的质粒pAN7-1通过限制性内切酶介导法转入植病生防真菌粉红粘帚霉Clonostachys rosea 67-1中,并由此建立该生防真菌的遗传转化体系。研究结果表明,PEG介质含量、限制性内切酶种类和浓度、质粒形态和反应时间对转化效率具有显著的影响。当质粒经线性化后加入40%PEG3350和20 U HindⅢ,室温下转化10 min时,转化效率可达到30～40 CFU.μg 1质粒DNA。PDA平板上连续培养3代,再转接到潮霉素抗性平板中,获得遗传稳定的转化子。PCR检测、Southern杂交和Real-time PCR检测等证明hph基因已整合到基因组中,且多为单拷贝插入。对随机挑取的50个转化子生长性状的测定结果表明,20%的转化子在PDA培养基中生长较快,12%的转化子产孢水平较高;另有16%～20%的转化子生长缓慢、产孢量减少;还有4%的转化子生长微弱,且无明显产孢现象。上述方法可以成功用于在粉红粘帚霉中引入外源基因,为今后生防粘帚霉高效工程菌株的构建提供了依据。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 HindlII 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 funsal soecies.

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 

分 类 号：S476.1[农业科学—农业昆虫与害虫防治]