Temasek Life Sciences Laboratory, 1 Research Link, Singapore.
Temasek Life Sciences Laboratory, 1 Research Link, Singapore; Department of Biological Sciences, National University of Singapore, Singapore; School of Biological Sciences, Nanyang Technological University, Singapore.
Fungal Genet Biol. 2014 Jul;68:71-6. doi: 10.1016/j.fgb.2014.04.005. Epub 2014 Apr 24.
A sulfonylurea-resistant allele of the ILV2 gene encoding an acetolactate synthase from the rice-blast fungus Magnaporthe oryzae has been extensively used in fungal transformation as a dominant selectable marker that confers resistance to chlorimuron ethyl. We devised a novel strategy for site-specific integration of foreign DNA via sulfonylurea resistance reconstitution (SRR) by replacing the native ILV2 with the sulfonylurea-resistant ILV2(SUR) variant. In contrast to random ectopic integration, SRR-based targeted incorporation at a defined locus eliminates position/orientation effects, unnecessary mutations and/or variation in gene expression. Independent transformants derived from the same SRR construct showed consistent and reproducible fluorescent signal in M. oryzae. Furthermore, the high frequency (>95%) of ILV2-specific targeted integration via SRR circumvents the need for a deficiency in non-homologous end joining (NHEJ) pathway in the recipient strain. Unlike the split-marker technique, which is particularly suitable for targeted gene replacement, the SRR strategy should prove useful for promoter analyses, gene tagging and/or complementation analyses in filamentous fungi.
一个编码来自稻瘟病菌（Magnaporthe oryzae）的乙酰乳酸合酶的 ILV2 基因的抗磺酰脲抗性等位基因已被广泛用于真菌转化，作为赋予对氯嘧磺隆乙基抗性的显性选择标记。我们设计了一种通过磺酰脲抗性重建（SRR）进行定点整合外源 DNA 的新策略，即用抗磺酰脲的 ILV2（SUR）变体取代天然的 ILV2。与随机异位整合相比，基于 SRR 的靶向整合在一个确定的基因座上消除了位置/取向效应、不必要的突变和/或基因表达的变化。来自相同 SRR 构建体的独立转化子在稻瘟病菌中显示出一致和可重复的荧光信号。此外，通过 SRR 进行 ILV2 特异性靶向整合的高频率（>95%）避免了受体菌株中非同源末端连接（NHEJ）途径的缺陷。与特别适用于靶向基因替换的分割标记技术不同，SRR 策略应该对丝状真菌中的启动子分析、基因标记和/或互补分析有用。
