Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Av. Prof. Lineu Prestes 748, 05508-000 São Paulo, SP, Brazil.
Fungal Genet Biol. 2011 Aug;48(8):806-11. doi: 10.1016/j.fgb.2011.02.006. Epub 2011 Mar 17.
Agrobacterium tumefaciens is widely used for plant DNA transformation and more recently, has also been used to transform yeast, filamentous fungi and even human cells. Using this technique, we developed the first transformation protocol for the saprobic aquatic fungus Blastocladiella emersonii, a Blastocladiomycete localized at the base of fungal phylogenetic tree, which has been shown as a promising and interesting model of study of cellular function and differentiation. We constructed binary T-DNA vectors containing hygromycin phosphotransferase (hph) or enhanced green fluorescent protein (egfp) genes, under the control of Aspergillus nidulans trpC promoter and terminator sequences. 24 h of co-cultivation in induction medium (IM) agar plates, followed by transfer to PYG-agar plates containing cefotaxim to kill Agrobacterium tumefsciens and hygromycin to select transformants, resulted in growth and sporulation of resistant transformants. Genomic DNA from the pool o resistant zoospores were shown to contain T-DNA insertion as evidenced by PCR amplification of hph gene. Using a similar protocol we could also evidence the expression of enhanced green fluorescent protein (EGFP) in zoospores derived from transformed cells. This protocol can also open new perspectives for other non-transformable closely related fungi, like the Chytridiomycete class.
根癌农杆菌被广泛用于植物 DNA 转化，最近也被用于转化酵母、丝状真菌，甚至人类细胞。利用这项技术，我们开发了第一个腐生水生真菌 Blastocladiella emersonii 的转化方案， Blastocladiella emersonii 是一种 Blastocladiomycetes 真菌，位于真菌系统发育树的基部，它是研究细胞功能和分化的一个很有前途和有趣的模型。我们构建了含有潮霉素磷酸转移酶（hph）或增强型绿色荧光蛋白（egfp）基因的二元 T-DNA 载体，受 Aspergillus nidulans trpC 启动子和终止子序列的控制。在诱导培养基（IM）琼脂平板上共培养 24 小时，然后转移到含有头孢噻肟的 PYG-琼脂平板上以杀死根癌农杆菌和潮霉素以选择转化体，结果抗性转化体生长和产孢。从抗生孢子的混合 DNA 中显示出 T-DNA 插入，这是通过 hph 基因的 PCR 扩增证明的。使用类似的方案，我们还可以证明从转化细胞衍生的游动孢子中表达增强型绿色荧光蛋白（EGFP）。该方案也可以为其他非可转化的密切相关的真菌（如壶菌纲）开辟新的前景。
