High reliability transformation of the basal fungus Mucor circinelloides by electroporation
High reliability transformation of the basal fungus Mucor circinelloides by electroporation
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DOI：
10.1016/j.mimet.2011.01.002
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发表时间：
2011-03-01
影响因子：
2.2
通讯作者：
Garre, Victorian
中科院分区：
文献类型：
--
作者：
Gutierrez, Adrian;Lopez-Garcia, Sergio;Garre, Victorian
研究卷枝毛霉生物学的分子方法主要依赖于基于聚乙二醇的转化方法的存在，这是在放线菌真菌中最有效的方法之一。然而，该方法的可靠性差和转化率低是许多生物过程的分子研究的主要障碍。本文介绍了一种简单可靠的变换M.电穿孔法获得的卷枝藻原生质体。25 μ F电容、400 Ω电阻和4 kV/cm场强的高压脉冲被认为是将DNA递送到M中的最佳电条件。卷须状原生质体。在这些电条件下，用几种自我复制质粒和菌株组合进行了成功的转化，产生高达500多个转化体/μ g DNA。还实现了转基因（贝氏不动杆菌的atfA基因）在特定基因座（carRP）中的靶向DNA整合。这种转化方法将大大促进这种真菌生物学的深入分子遗传学研究。(C)2011 Elsevier B. V.保留所有权利。
Molecular approaches to study the biology of the zygomycete Mucor circinelloides depend mainly on the existence of a polyethylene glycol-based transformation method, which is one of the most efficient in zygomycete fungi. However, the poor reliability and low transformation rates of this method are major obstacles in the molecular study of a number of biological processes. This paper describes an easy and reliable method to transform M. circinelloides protoplasts by electroporation. A high-voltage pulse of 25 mu F capacitance, 400 Omega resistance, and 4 kV/cm field strength were seen to be the optimal electrical conditions for delivering DNA into M. circinelloides protoplasts. Under these electrical conditions, successful transformations were carried out with several self-replicative plasmid and strain combinations, producing up to more than 500 transformants per mu g DNA. Targeted DNA integration of a transgene (atfA gene of Acinetobacter baylyi) in a particular locus (carRP) was also achieved. This transformation method will considerably facilitate in-depth molecular genetic studies of the biology of this fungus. (C) 2011 Elsevier B.V. All rights reserved.