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文章摘要
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
投稿时间:2018-07-13  修订日期:2018-09-17
DOI:
中文关键词: 农杆菌转化  双元载体  黑曲霉  脂肪酶  条件优化
英文关键词: Agrobacterium-mediated transformation  binary vector  Aspergillus niger  lipase  optimization
基金项目:国家自然科学基金项目(面上项目,重点项目,重大项目),
作者单位E-mail
朱思远 江南大学 zsy934330940@163.com 
徐岩 江南大学  
喻晓蔚 江南大学 bioyuxw@aliyun.com 
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中文摘要:
      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200 µM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。
英文摘要:
      The method for Agrobacterium-mediated transformation of Aspergillus niger was improved by optimizing the co-culture materials, Agrobacterium species, inducer concentration, co-culture time, co-culture temperature as well as the Agrobacterium concentration during co-culture. The optimum conditions for the transformation of A. niger are 107/ml non-germinated fresh spores and OD600 of Agrobacterium cultured to 0.9-1.0 in a ratio of 1:1, and the IM plate with acetosyringone concentration of 200 μM. 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.
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