Yu Jae-Hyuk, Hamari Zsuzsanna, Han Kap-Hoon, Seo Jeong-Ah, Reyes-Domínguez Yazmid, Scazzocchio Claudio
Department of Food Microbiology and Toxicology, The University of Wisconsin, Madison, WI 53706, USA.
Fungal Genet Biol. 2004 Nov;41(11):973-81. doi: 10.1016/j.fgb.2004.08.001.
Gene replacement via homologous double crossover in filamentous fungi requires relatively long (preferentially >0.5 kb) flanking regions of the target gene. For this reason, gene replacement cassettes are usually constructed through multiple cloning steps. To facilitate gene function studies in filamentous fungi avoiding tedious cloning steps, we have developed a PCR-assisted DNA assembly procedure and applied it to delete genes in filamentous fungi. While the principle of this procedure is essentially the same as other recently reported PCR-based tools, our technique has been effectively used to delete 31 genes in three fungal species. Moreover, this PCR-based method was used to fuse more than 10 genes to a controllable promoter. In this report, a detailed protocol for this easy to follow procedure and examples of genes deleted or over-expressed are presented. In conjunction with the availability of genome sequences, the application of this technique should facilitate functional characterization of genes in filamentous fungi. To stream line the analysis of the transformants a relatively simple procedure for genomic DNA or total RNA isolation achieving approximately 100 samples/person/day is also presented.
在丝状真菌中，通过同源双交换进行基因替换需要目标基因相对较长（最好>0.5 kb）的侧翼区域。因此，基因替换盒通常通过多个克隆步骤构建。为了便于在丝状真菌中进行基因功能研究，避免繁琐的克隆步骤，我们开发了一种PCR辅助的DNA组装程序，并将其应用于丝状真菌中的基因删除。虽然该程序的原理与最近报道的其他基于PCR的工具基本相同，但我们的技术已有效地用于删除三种真菌物种中的31个基因。此外，这种基于PCR的方法被用于将10多个基因融合到一个可控启动子上。在本报告中，给出了这个易于遵循的程序的详细方案，以及基因删除或过表达的实例。结合基因组序列的可用性，该技术的应用应有助于丝状真菌中基因的功能表征。为了简化转化体的分析，还介绍了一种相对简单的基因组DNA或总RNA分离程序，每人每天可实现约100个样本。
