A dual selection based, targeted gene replacement tool for Magnaporthe grisea and Fusarium oxysporum
A dual selection based, targeted gene replacement tool for Magnaporthe grisea and Fusarium oxysporum
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DOI：
10.1016/j.fgb.2005.03.004
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发表时间：
2005-06-01
影响因子：
3
通讯作者：
Kang, SC
中科院分区：
文献类型：
--
作者：
Khang, CH;Park, SY;Kang, SC
真菌基因组测序的快速发展为真菌生物学功能基因组分析提供了许多新的机会，通过系统诱变测序鉴定的基因。然而，缺乏有效的靶向基因替换工具是真菌功能基因组学的一个限制因素，因为它通常需要筛选大量的转化子来识别所需的突变体。本研究以稻瘟病菌Magnaporthe grisea和oxysportan Fusarium为研究对象，建立了一种高效的基因替换方法，并对影响该方法效率的因素进行了评价。该方法基于农杆菌介导的转化，将单纯疱疹病毒胸苷激酶（HSVtk）基因两侧的靶基因突变等位基因作为对抗异位转化体的条件阴性选择标记。HSVtk基因产物将5-氟-2'-脱氧尿苷转化为一种对多种真菌有毒的化合物。由于异位转化体表达HSVtk，而基因替代突变体缺乏HSVtk，因此在含有5-氟-2'-脱氧尿苷修饰的培养基上培养转化体有助于通过反选择异位转化体来识别目标突变体。除了稻瘟病菌和oxysporum外，该方法和相关载体可能适用于广泛的真菌基因操作，因此有可能作为一种有效的、通用的功能基因组工具，利用真菌基因组序列数据来研究真菌生物学。(c) 2005爱思唯尔公司版权所有。
Rapid progress in fungal genome sequencing presents many new opportunities for functional genomic analysis of fungal biology through the systematic mutagenesis of the genes identified through sequencing. However, the lack of efficient tools for targeted gene replacement is a limiting factor for fungal functional genomics, as it often necessitates the screening of a large number of transformants to identify the desired mutant. We developed an efficient method of gene replacement and evaluated factors affecting the efficiency of this method using two plant pathogenic fungi, Magnaporthe grisea and Fusarium oxysportan. This method is based on Agrobacterium tumefaciens-mediated transformation with a mutant allele of the target gene flanked by the herpes simplex virus thymidine kinase (HSVtk) gene as a conditional negative selection marker against ectopic transformants. The HSVtk gene product converts 5-fluoro-2'-deoxyuridine to a compound toxic to diverse fungi. Because ectopic transformants express HSVtk, while gene replacement mutants lack HSVtk, growing transformants on a medium amended with 5-fluoro-2'-deoxyuridine facilitates the identification of targeted mutants by counter-selecting against ectopic transformants. In addition to M. grisea and F oxysporum, the method and associated vectors are likely to be applicable to manipulating genes in a broad spectrum of fungi, thus potentially serving as an efficient, universal functional genomic tool for harnessing the growing body of fungal genome sequence data to study fungal biology. (c) 2005 Elsevier Inc. All rights reserved.