摘要
采用根癌农杆菌介导的方法,成功地建立了丝状生防真菌深绿木霉(Trichoderma atroviride)菌株T23的遗传转化体系。并且,通过提高筛选培养基中潮霉素B的浓度和调整农杆菌的培养时间,对转化体系作了进一步的优化。转化效率约为50个突变体/107个分生孢子。所有转化子经继代培养5代,潮霉素B抗性筛选后,共得到118个遗传稳定的转化子。随机抽取部分转化子,进行PCR和Southern blot分子鉴定,结果证实外源的T-DNA已经随机整合到T23的基因组中。通过形态学观察,筛选出3个在产孢量和菌丝体生长速度方面显著不同于野生型菌株T23的转化子。农杆菌介导的遗传转化方法在深绿木霉菌株T23上的成功运用,将为研究该菌的功能基因组提供强有力的工具。
Agrobacterium tumefociens-mediated transformation （ATMT） was applied in Trichoderma atroviride strain T23. Meanwhile, the transformation method was optimized by adjusting the concentration of hygromycin B in selection medium and exploring the incubation time of Agrobacterial AGL-1 cells. The transformation efficiency was about 50 transformants per 10^7 conidia. All transformants were successively subcuhured for five generations, and then screened on selective plates, 118 genetically stable transformants were obtained. Some of them were randomly selected to conduct PCR and Southern blotting analysis, and the result showed that all transformants tested were arbitrarily integrated with exogenous T-DNA in genome. Compared to wild-type strain T23, three transformants were obviously different in spore production and mycelia growth rate. The successful application of ATMT technique in T. atroviride T23 will facilitate the study of functional genome of this organism.
出处
《上海交通大学学报（农业科学版）》
2009年第5期489-493,共5页
Journal of Shanghai Jiaotong University(Agricultural Science)
基金
上海市青年科技启明星计划项目(07QA14035)
国家高技术研究与发展计划项目(2006AA10Z409
2006AA10A211)
上海交通大学第14期PRP项目(T15014045)
关键词
根癌农杆菌
深绿木霉
转化子
遗传稳定性
潮霉素B抗性
Agrobacterium tumefaciens
Trichoderma atroviride
transformants
genetic stability
hygromycin B resistance
