根癌农杆菌介导的丝状真菌简青霉遗传转化的研究.pdf

37 10 ( ) Vol. 37, No. 10 2 0 1 0 1 0 Journal of Hunan University( Natural Sciences) Oct 2 0 1 0 : 1 742974( 2010) * 朱咏华, 吴 康, 郑 芒, 靳 冉, 廖红东, 刘选明 ( , , 4 10082) : 利用根癌农杆菌LBA4404 介导, 建立了丝状真菌简 霉( P enici ll i um s i mp l i ciss i mum ) H5 的遗传转化系统. 潮霉素抗性筛选PCR 和Southern blot 分子鉴定等结果表 明: 筛选获得的转化子能够稳定遗传, 外源的TDNA 以单拷贝随机整合到简 霉的基因组 中. 实验初步研究了农杆菌浓度乙酰 丁香酮浓度和共培养时间等因素对转化效率的影响, 经过优化后转化体系的效率可达50 个转化子/ 105 个孢子. 农杆菌介导的遗传转化方法在 简 霉上的运用, 将为研究该菌的基因工程改造提供强有力的工具. : 根癌农杆菌; 简 霉; 转化 : Q933 : A A g r obacter ium T umef ac iensmediated Transformation of the Penicillium Simplicissimum H5 ZHU Yonghua,WU Kang, ZHENG Mang, JIN Ran,LIAO Hongdong, LIU Xuanming ( State Key Laboratory of Chemical Biosensing and Chemometrics, School of Biology, Hunan Univ, Changsha, Hunan 410082, China) Abstract:A g r obact eri um tumef aciensmediated transformation ( AT MT ) w as applied in Pe nici ll i um s i mp l iciss i mum H5 by A g robac ter i um tumef aciens LBA4404. T he results of hygromycinresistance selec tion, PCR and Southern blot analysis show ed that TDNA w as randomly inserted into the transformants w ith single copy and the transformants indicated genetic stability. T he transformation efficiency w as about 5 50 transformants per 10 spores by optimizing the coculture time, the concentration of A g r obact er ium t u mef ac ien and acetoyringone ( AS) . T he application of AT MT technique in Pe nici ll i um s imp licissi mum H5 w ill provide a pow erful tool for the genetic engineering of this fungus. Key words:ag robact er ium tumef aciens ; p enici l li um s i mp l ic iss i mum ; transfor