农杆菌介导转化黑曲霉条件优化及脂肪酶表达

doi:10.3969/j.issn.1673-1689.2020.05.008

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农杆菌介导转化黑曲霉条件优化及脂肪酶表达
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                        10.3969/j.issn.1673-1689.2020.05.008
                    
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Optimization of Agrobacterium tumefaciens-Mediated Transformation of Aspergillus niger and Expression of Lipase

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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200 μmol/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。

Abstract:

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 OD₆₀₀ 0.9~1.0 in a ratio of 1∶1, and 200 μmol/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.

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朱思远,徐岩,喻晓蔚.农杆菌介导转化黑曲霉条件优化及脂肪酶表达[J].食品与生物技术学报,2020,39(5):51-58.

ZHU Siyuan, XU Yan, YU Xiaowei. Optimization of Agrobacterium tumefaciens-Mediated Transformation of Aspergillus niger and Expression of Lipase[J]. Journal of Food Science and Biotechnology,2020,39(5):51-58.

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在线发布日期: 2020-06-19
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