A High-Throughput Gene Disruption Methodology for the Entomopathogenic Fungus Metarhizium robertsii
A High-Throughput Gene Disruption Methodology for the Entomopathogenic Fungus Metarhizium robertsii
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昆虫病原真菌绿僵菌的高通量基因破坏方法
DOI：
10.1371/journal.pone.0107657
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发表时间：
2014-09-15
期刊：
影响因子：
3.7
通讯作者：
Fang, Weiguo
中科院分区：
文献类型：
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作者：
Xu, Chuan;Zhang, Xing;Fang, Weiguo
系统的基因破坏是一种直接的方法来询问真菌基因组，以功能表征参与各种生物过程的全套基因。罗伯特绿僵菌是非常多才多艺的，它是节肢动物的病原体，腐生植物和根际有益的定植者。因此，M. robertsii可以作为代表，同时研究几种“模式”真菌Saccharomyces cerevisiae和Neurospora crassa所没有的主要生活方式；对robertsii进行系统的遗传分析将有助于其他真菌的研究。为了系统地破坏罗伯特氏杆菌的基因，我们开发了一种高通量基因破坏方法，其中包括两种技术。一种是改良的基于oscar的高通量基因破坏质粒构建。该技术涉及两个供体质粒（含有抗除草剂基因Bar的pA-Bar-OSCAR和含有另一种抗除草剂基因Sur的pA-Sur-OSCAR）和一个受体二元质粒pPK2-OSCAR-GFP，该质粒是通过将pPK2-bar-GFP中的Bar盒替换为ccdB盒和重组识别位点而产生的。利用该技术，可以在两天内一步克隆出基因破坏质粒。另一种是以Ku70缺失突变体（ΔMrKu70）为受体菌株，基于同源重组的高效基因破坏技术。MrKu70是一种编码真菌非同源末端连接DNA修复关键成分的基因，其缺失可显著提高基因破坏效率。在ΔMrKu70中破坏条件相关基因Cag8的频率为93%，而在野生型菌株中为7%。由于ΔMrKu70在发育、致病性和对各种非生物胁迫的耐受性方面与野生型菌株没有什么不同，因此它可以作为受体菌株，用于系统的基因破坏项目，以表征robertsii m.s生物过程中涉及的整套基因。
Systematic gene disruption is a direct way to interrogate a fungal genome to functionally characterize the full suite of genes involved in various biological processes. Metarhizium robertsii is extraordinarily versatile, and it is a pathogen of arthropods, a saprophyte and a beneficial colonizer of rhizospheres. Thus, M. robertsii can be used as a representative to simultaneously study several major lifestyles that are not shared by the “model” fungi Saccharomyces cerevisiae and Neurospora crassa; a systematic genetic analysis of M. robertsii will benefit studies in other fungi. In order to systematically disrupt genes in M. robertsii, we developed a high-throughput gene disruption methodology, which includes two technologies. One is the modified OSCAR-based, high-throughput construction of gene disruption plasmids. This technology involves two donor plasmids (pA-Bar-OSCAR with the herbicide resistance genes Bar and pA-Sur-OSCAR with another herbicide resistance gene Sur) and a recipient binary plasmid pPK2-OSCAR-GFP that was produced by replacing the Bar cassette in pPK2-bar-GFP with a ccdB cassette and recombination recognition sites. Using this technology, a gene disruption plasmid can be constructed in one cloning step in two days. The other is a highly efficient gene disruption technology based on homologous recombination using a Ku70 deletion mutant (ΔMrKu70) as the recipient strain. The deletion of MrKu70, a gene encoding a key component involved in nonhomologous end-joining DNA repair in fungi, dramatically increases the gene disruption efficiency. The frequency of disrupting the conidiation-associated gene Cag8 in ΔMrKu70 was 93% compared to 7% in the wild-type strain. Since ΔMrKu70 is not different from the wild-type strain in development, pathogenicity and tolerance to various abiotic stresses, it can be used as a recipient strain for a systematic gene disruption project to characterize the whole suite of genes involved in the biological processes of M. robertsii.