A novel method used to delete a new Aspergillus fumigatus ABC transporter-encoding gene
A novel method used to delete a new Aspergillus fumigatus ABC transporter-encoding gene
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DOI：
10.1007/s00294-002-0313-z
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发表时间：
2002-07-01
期刊：
影响因子：
2.5
通讯作者：
Brakhage, AA
中科院分区：
文献类型：
--
作者：
Langfelder, K;Gattung, S;Brakhage, AA
烟曲霉是一种重要的机会性人类致病真菌。在免疫功能严重受损的患者中，真菌会导致危及生命的疾病，例如肺炎和侵袭性曲霉菌病。为了更好地了解烟曲霉毒力的关键要素并确定可能的药物靶标，必须能够产生基因缺失菌株。直到最近，可用的分子技术还没有提供基因删除的快速方法。针对构巢曲霉描述的新方法适用于烟曲霉。该方法速度快并且同源重组效率提高。通过使用表达 lambda 红色操纵子的大肠杆菌菌株，可以诱导侧翼 > 50 bp 区域的 PCR 片段与含有目的基因的粘粒发生体内重组。这会产生粘粒，其中感兴趣的基因已被双功能标记取代。这种粘粒在此处使用的烟曲霉的选择标记pyrG周围具有大的侧翼区域，这导致烟曲霉的高重组效率。在这里，我们在烟曲霉中鉴定了一个新的 ABC 转运蛋白编码基因，命名为 abcA。通过使用这种方法，产生了烟曲霉敲除突变体，这提供了证据表明这种产生基因缺失的方法也可以用于烟曲霉，并显着拓宽了我们研究烟曲霉的分子技术库。
Aspergillus fumigatus is an important opportunistic human pathogenic fungus. In severely immunocompromised patients, the fungus causes life-threatening diseases, such as pneumonia and invasive aspergillosis. In order to obtain a better understanding of the key elements involved in A. fumigatus virulence and for identifying possible drug targets, it is essential to be able to generate gene-deletion strains. Until recently, the molecular techniques available did not provide a rapid method for gene deletion. A novel method described for A. nidulans was adapted for A. fumigatus. This method is quick and produces an increased homologous recombination efficiency. By using an Escherichia coli strain expressing the lambda red operon, it is possible to induce an in vivo recombination of a PCR fragment flanked by >50-bp regions with a cosmid containing the gene of interest. This produces cosmids in which the gene of interest has been replaced by a bifunctional marker. Such cosmids have large flanking regions surrounding the selectable marker pyrG of A. fumigatus used here, which result in high recombination efficiencies in A. fumigatus. Here, we identified a new ABC transporter-encoding gene in A. fumigatus, designated abcA. By using this method, an A. fumigatus knock-out mutant was generated, providing evidence that this method of generating gene deletions can also be used in A. fumigatus and significantly broadens our repertoire of molecular techniques to study A. fumigatus.