根癌农杆菌介导的烟曲霉转化条件的优化  

Optimization of factors affecting genetic transformation of aspergillus fumigatus via Agrobacterium tumefaciens

作　　者：龙朝钦[1] 邓军[1] 郝飞[1] 钟白玉[1] 周村建[1] 

机构地区：[1]第三军医大学西南医院皮肤科,重庆400038

出　　处：《西部医学》2008年第2期261-264,共4页Medical Journal of West China

摘　　要：目的探讨根癌农杆菌介导烟曲霉的遗传转化及其影响因素。方法构建根癌农杆菌二元载体PDHt/SK,二元载体转化根癌农杆菌,分别对共培养温度(23、24、25、26、27、28)℃、媒介(尼龙膜、硝化纤维膜、液相静置培养)、共培养时间(12、24、36、48h)、预培养加乙酰丁香酮与否、加入乙酰丁香酮的浓度(100μg/ml、200μg/ml、300μg/ml、400μg/ml);分生孢子与根癌农杆菌的不同比例(1∶1、1∶10、1∶100、1∶1000)进行根癌农杆菌介导烟曲霉的转化,观察上述条件下的根癌农杆菌介导的烟曲霉转化效率,用潮霉素抗性基因筛选转化子。结果共培养温度、转化媒介、共培养时间、共培养加入乙酰丁香酮不同的浓度、分生孢子与根癌农杆菌的不同比例均对根癌农杆菌介导烟曲霉转化效率产生影响,但是预培养加乙酰丁香酮与否对根癌农杆菌转化烟曲霉的效率影响不明显。结论根癌农杆菌可成功介导烟曲霉的遗传转化,其转化过程受诸多因素的影响。Objective To discuss the factors affecting genetic transformation of aspergillus fumigatus via Agrobacterium tumefaciens. Methods We constructed binary vector pDHt/SK of Agrobacterium tumefaciens. The Agrobacterium tumefaciens-mediated transformation （ATMT） was conducted at different co-cultivation temperature （23, 24, 25, 26, 27, 28℃ ） ,different transformat media （nylon, nitrocellulose filters, liquid phase cultivation） ,different co-cultivation period（12, 24, 36, 48h）,AS preincubation or not,different concentrations of AS in co-cultivation（100μg/ ml, 200μg/ml, 300μg/ml, 400μg/ml）,co-cultivation at different ratios of conidia to bacteria（1 ： 1, 1 ： 10, 1 ： 100, 1 ： 1000）, to explore the tansformation efficiency. Hygromycin resistance cassette was used to select the positive transformant. Results ATMT was affected by co-cultivatlon temperature,media, co-cultivation period, concentration of AS in co-cultivation,co-cultivation at different ratios of conidia to bacteria. No matter AS preincubation or not, it failed to show a noticeable impact on ATMT. Conclusion This method can mediate genetic transformation of aspergillus fumigatus via Agrobacterium tumefaciens successfully but affected by many factors in this process.

关 键 词：烟曲霉 根癌农杆菌 潮霉素抗性 

分 类 号：R379[医药卫生—病原生物学]