丝状真菌中一个可选择标记的基因替换。

Nalleli Garcia, Alexa N Farmer, Richmond Baptiste, Jessie Fernandez
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摘要

稻瘟病菌是一种丝状真菌,对全世界水稻作物造成有害的稻瘟病。多年来,m.o ryzae由于其已测序的基因组,对功能遗传学的适应性以及在实验室环境中跟踪的能力而成为研究植物病原体相互作用的优秀模式生物。因此,对m.o ryzae进行基因删除的技术范围从通过CRISPR-Cas9进行基因组编辑到通过同源重组进行基因替换。该方案侧重于详细介绍如何通过分裂标记方法在模式生物M. oryzae中进行基因替换。这种技术依赖于用一个对特定可选化学物质具有抗性的基因取代感兴趣基因的开放阅读框,破坏感兴趣基因的转录,产生一个敲除突变的m.o ryzae菌株。引物设计,peg介导的原生质体转化和真菌DNA提取筛选的全面概述。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

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Gene Replacement by a Selectable Marker in the Filamentous FungusMagnaporthe oryzae.

Magnaporthe oryzaeis a filamentous fungus responsible for the detrimental rice blast disease afflicting rice crops worldwide. For years, M. oryzae has served as an excellent model organism to study plant pathogen interactions due to its sequenced genome, its amenability to functional genetics, and its capacity to be tracked in laboratory settings. As such, techniques to genetically manipulate M. oryzae for gene deletion range from genome editing via CRISPR-Cas9 to gene replacement through homologous recombination. This protocol focuses on detailing how to perform gene replacement in the model organism, M. oryzae, through a split marker method. This technique relies on replacing the open reading frame of a gene of interest with a gene conferring resistance to a specific selectable chemical, disrupting the transcription of the gene of interest and generating a knockout mutant M. oryzae strain. Key features Comprehensive overview of primer design, PEG-mediated protoplast transformation, and fungal DNA extraction for screening.

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