根癌农杆菌介导里氏木霉转化体系的建立和条件优化  

Agrobacterium tumefaciens Mediated Transformation of Trichoderma reesei and Optimization of the Transforming Conditions

作　　者：刘刚[1] 杨长得[1] 康康[1] 邢苗[1] 田生礼[1] 

机构地区：[1]深圳市微生物基因工程重点实验室深圳大学生命科学学院,广东深圳518060

出　　处：《生物技术》2011年第4期44-48,共5页Biotechnology

基　　金：国家自然科学基金项目(No.31070044);深圳市科技计划项目(No.JC201005280559A;JC201005250041A)资助

摘　　要：目的:采用根癌农杆菌介导的转化方法实现丝状真菌里氏木霉的遗传转化,并优化转化条件。方法:构建含潮霉素抗性基因(hph)的双元载体pCAM-hph后,转化根癌农杆菌LBA4404获得转化菌株。将根癌农杆菌的转化菌株和里氏木霉的分生孢子共培养后在含100μg/mL潮霉素的抗性平板上筛选里氏木霉转化子,并采用PCR扩增和序列测定对转化子中的插入片段进行了分析。结果:使用根癌农杆菌介导的转化方法转化里氏木霉,每106个分生孢子可获得25.8个转化子。最佳的转化条件为:农杆菌初始浓度为OD660约为0.8,孢子数为106个,共培养时间为48h,pH为5.0～5.5,培养温度为28℃。结论:建立了根癌农杆菌介导的里氏木霉转化方法,并获得了最佳的转化条件。Objective：Transformation of the filamentous fungus Trichoderma reesei was achieved using Agrobacterium tumeficiens-mediated transformation（ATMT） method,and the factors influencing transformation efficiency were evaluated.Method：The binary vector pCAM-hph containing the hygromycin phosphotransferase（hph） gene was constructed based on a commercial vector pCAMBIA 0380,and was transformed into A.tumeficiens LBA4404.The A.tumeficiens transformants were co-cultivated with the T.reesei conidial spores,and the fungal transformants were selected on PDA plate containing 100μg/mL hygromycin.The DNA fragment that was inserted into the fungal transformants was analyzed through PCR amplification and DNA sequencing.Result：ATMT method was effectively applied in transformation of T.reesei,with a transformation efficiency of obtaining 25.8 transfotmats per 106 conidial spores.Acetosyringone concentration,the concentrations of the bacterial cells and the conidial spores,period of co-cultivation,and pH of the medium in co-cultivation evidently affected the efficiency of transformation.The optimal transformation conditions were： the concentration of the bacterial cells was OD660 0.8,the concentration of conidial spores was 106/ml,the co-cultivation period was 48 hours,the pH of co-cultivation was 5.0~5.5,and the culture temperature was 28℃.Conclusion：The ATMT method for transformation of T.reesei was constructed,and the transformation condition was optimized.

关 键 词：里氏木霉 根癌农杆菌 分生孢子 潮霉素磷酸转移酶 双元载体 

分 类 号：Q786[生物学—分子生物学]