Schilde C, Wöstemeyer J, Burmester A
Friedrich-Schiller-Universität Jena, Institut für Mikrobiologie, Lehrstuhl für Allgemeine Mikrobiologie und Mikrobengenetik, Germany.
Arch Microbiol. 2001 Jan;175(1):1-7. doi: 10.1007/s002030000228.
Mucoralean fungi (Zygomycota) are used for many industrial processes and also as important model organisms for investigating basic biological problems. Their genetic analysis is severely hampered by low transformation frequencies, by their strong tendency towards autonomous replication of plasmids instead of stable integration, and by the lack of reliable genetic reporter systems. We constructed plasmids for transforming the model zygomycete Absidia glauca that carry the versatile reporter gene coding for green fluorescent protein (GFP). gfp expression is controlled either by the homologous actin promoter or the promoter for the elongation factor of translation, EF1alpha. These plasmids also confer neomycin resistance and carry one of two genetic elements (rag1, seg1) that improve mitotic stability of the plasmid. The gfp constructs were replicated extrachromosomally and could be recovered from retransformed Escherichia coli cells. gfp expression was monitored by epifluorescence microscopy. The gfp reporter gene plasmids presented here for the model zygomycete A. glauca constitute the first reliable system that allows the monitoring of gene expression in this important group of fungi.
毛霉目真菌（接合菌门）被用于许多工业过程，并且作为研究基本生物学问题的重要模式生物。它们的遗传分析受到低转化频率、质粒自主复制而非稳定整合的强烈倾向以及缺乏可靠的遗传报告系统的严重阻碍。我们构建了用于转化模式接合菌灰绿犁头霉的质粒，这些质粒携带编码绿色荧光蛋白（GFP）的通用报告基因。gfp的表达由同源肌动蛋白启动子或翻译延伸因子EF1α的启动子控制。这些质粒还赋予新霉素抗性，并携带两种遗传元件（rag1、seg1）之一，可提高质粒的有丝分裂稳定性。gfp构建体在染色体外复制，并且可以从重新转化的大肠杆菌细胞中回收。通过落射荧光显微镜监测gfp的表达。这里展示的用于模式接合菌灰绿犁头霉的gfp报告基因质粒构成了第一个可靠的系统，可用于监测这一重要真菌类群中的基因表达。
