农杆菌介导转化黑曲霉条件优化及脂肪酶表达  

Optimization of Agrobacterium tumefaciens-Mediated Transformation of Aspergillus niger and Expression of Lipase

作　　者：朱思远 徐岩 喻晓蔚[1,2,3] ZHU Siyuan;XU Yan;YU Xiaowei(School of Biotechnology,Jiangnan University,Wuxi 214122,China;Key Laboratory of Industrial Biotechnology,Ministry of Education,Jiangnan University,Wuxi 214122,China;Industrial Technology Research Institute of Jiangnan University,Suqian 223814,China)

机构地区：[1]江南大学生物工程学院,江苏无锡214122 [2]江南大学工业生物技术教育部重点实验室,江苏无锡214122 [3]宿迁市江南大学产业技术研究院,江苏宿迁223814

出　　处：《食品与生物技术学报》2020年第5期51-58,共8页Journal of Food Science and Biotechnology

基　　金：国家自然科学基金面上项目(31671799);江苏省“六大人才高峰”计划项目(NY-010);江苏高校品牌专业建设工程资助项目。

摘　　要：优化了农杆菌介导转化黑曲霉的方法,优化条件包括共培养材料、农杆菌种类、诱导剂浓度、共培养时间、共培养温度以及共培养时农杆菌的菌体浓度。结果表明,农杆菌介导转化黑曲霉的最适条件为107个/mL不萌发的新鲜孢子与OD600培养至0.9~1.0的农杆菌以1∶1的比例混合后,在乙酰丁香酮浓度为200μmol/L的IM平板上,23℃避光培养48 h后进行转膜,转化子个数可达到(60±5)个转化子/106个孢子,且阳性率达到90%以上。并且构建了同源黑曲霉脂肪酶的组成型和诱导型启动子表达载体,通过优化后的农杆菌介导转化方法转化至黑曲霉中,利用罗丹明橄榄油平板对产酶转化子进行筛选鉴定,并获得了阳性克隆。The method for Agrobacterium-mediated transformation of Aspergillus niger was improved by optimizing the co-culuture materials,Agrobacterium species,inducer concentration,co-culture time and temperature as well as the Agrobacterium concentration during co-culture.The optimum conditions for the transformation of A.niger were 107/mL non-germinated fresh spores and Agrobacterium with OD6000.9~1.0 in a ratio of 1∶1,and 200μmol/L acetosyringone in the IM plate.The mixed cells were then transfected at 23℃for 48 h,and the number of transformants reached 60±5 transformants/106 spores,and the positive rate reached over 90%.The expression vector of homologous A.niger lipase with constitutive or inducible promoter was constructed and transformed into A.niger by the optimized Agrobacterium-mediated transformation method.Finally the positive transformants were identified by Rhodamine-olive oil plate.

关 键 词：农杆菌转化 双元载体 黑曲霉 脂肪酶 优化 

分 类 号：Q785[生物学—分子生物学]