Faculty of Health Sciences, University of Macau, Avenida da Universidade, Taipa, Macau.
Faculty of Health Sciences, University of Macau, Avenida da Universidade, Taipa, Macau; Institute of Translational Medicine, Faculty of Health Sciences, University of Macau, Avenida da Universidade, Taipa, Macau.
Fungal Genet Biol. 2019 Apr;125:28-35. doi: 10.1016/j.fgb.2019.01.004. Epub 2019 Jan 11.
Gene manipulation is an important routine technique and its efficiency is often a rate-limiting step in research. To facilitate gene manipulation in filamentous fungi, we adapted the S. cerevisiae Gene Deletion and Gene Tagging plasmid collections to include additional selectable markers that make the useful resources applicable to other fungi. Three markers for auxotrophic selection in Aspergillus and related species (the riboB, pyroA and pyrG genes of Aspergillus fumigatus) and a dominant selectable marker for glufosinate resistance (the Bar gene from Streptomyces hygroscopicus) were introduced to the collections. A total of fifty-six plasmids were constructed for all combinations between the four new markers and thirteen epitope tags (viz., 3xHA, 13xMYC, 3xFLAG, FLAG, MYC, T7, HIS, Strep, S, HSV, VSV-G, V5 and GFP). The selectable marker and epitope tag cassettes are positioned between two universal sequences in the plasmids, and therefore, can be amplified by PCR using the same pair of primers. With these plasmids, we have also established a simple and efficient procedure for making gene deletion and gene tagging transformation DNA constructs. The procedure, along with the universal flanking sequences, allows quick and easy interchange of selectable marker and epitope cassettes in transformation DNA constructs for different selection and/or tagging. To demonstrate utility and efficiency of the system, we simultaneously performed C-terminal tagging of HapB - a subunit of the highly conserved Aspergillus nidulans CCAAT binding complex that plays important transcriptional regulatory roles - using ten different epitopes in order to identify those neutral to HapB function in vivo. It is expected that the expanded plasmid collections coupled with the simple construction strategy would facilitate gene manipulation in many fungal species.
基因操作是一种重要的常规技术，其效率通常是研究中的一个限速步骤。为了促进丝状真菌中的基因操作，我们对酿酒酵母基因缺失和基因标记质粒文库进行了改造，增加了其他可选标记，使这些有用的资源适用于其他真菌。文库中引入了三个用于营养缺陷型选择的标记（烟曲霉的 riboB、pyroA 和 pyrG 基因）和一个用于草甘膦抗性的显性选择标记（吸水链霉菌的 Bar 基因）。总共构建了 56 个质粒，用于四个新标记和 13 个表位标签（即 3xHA、13xMYC、3xFLAG、FLAG、MYC、T7、HIS、Strep、S、HSV、VSV-G、V5 和 GFP）之间的所有组合。选择标记和表位标签盒位于质粒中的两个通用序列之间，因此可以使用相同的一对引物通过 PCR 进行扩增。有了这些质粒，我们还建立了一种简单高效的基因缺失和基因标记转化 DNA 构建程序。该程序与通用侧翼序列一起，允许在不同选择和/或标记的转化 DNA 构建体中快速、轻松地交换选择标记和表位盒。为了证明该系统的实用性和效率，我们同时使用 10 种不同的表位对高度保守的构巢曲霉 CCAAT 结合复合物的 HapB亚基进行 C 末端标记，以鉴定那些对 HapB 功能无影响的表位。预计扩展的质粒文库结合简单的构建策略将有助于许多真菌物种的基因操作。
