Gene disruption in Cryptococcus neoformans and Cryptococcus gattii by in vitro transposition
Gene disruption in Cryptococcus neoformans and Cryptococcus gattii by in vitro transposition
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DOI：
10.1007/s00294-005-0054-x
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发表时间：
2006-05-01
期刊：
影响因子：
2.5
通讯作者：
Kronstad, JW
中科院分区：
文献类型：
--
作者：
Hu, GG;Kronstad, JW
新型隐球菌和格特隐球菌是担子菌真菌，可感染免疫功能低下和免疫功能正常的人群。我们基于体外转座为这些物种开发了一种插入诱变策略，并通过破坏新型隐球菌菌株中的 URA5 基因和三个格特隐球菌菌株中的 CAP10 基因来测试该方法。我们针对 C. gatti 菌株 WM276 的鸟枪测序项目的基因组文库中含有 URA5 基因的质粒 DNA 或含有 CAP10 基因的质粒 DNA 进行靶向。在后一种情况下，来自组装的基因组序列的克隆的末端序列的可用性允许快速选择用于破坏的目标基因。通过体外转座将含有诺尔丝菌素 (NAT) 或新霉素 (Neo) 抗性盒的修饰转座子随机插入到靶 DNA 中。破坏的基因用于基因枪转化，随后通过 PCR 和 Southern blot 分析证实同源整合。这些结果表明，新兴的基因组资源与鸟枪法测序文库或克隆 PCR 产品的体外转座质粒 DNA 相结合，将促进隐球菌属物种的高通量遗传分析。
Cryptococcus neoformans and Cryptococcus gattii are basidiomycetous fungi that infect immunocompromised and immunocompetent people. We developed an insertional mutagenesis strategy for these species based on in vitro transposition and we tested the method by disrupting the URA5 gene in a strain of C. neoformans and the CAP10 gene in three strains of C. gattii. We targeted plasmid DNA containing the URA5 gene or plasmid DNA containing the CAP10 gene from genomic libraries from the shotgun sequencing project for the C. gatti strain WM276. In the latter case, the availability of the end sequences of the clones from the assembled genomic sequence allows rapid selection of target genes for disruption. Modified transposons containing the nourseothricin (NAT) or neomycin (Neo) resistance cassettes were randomly inserted into the target DNA by in vitro transposition. The disrupted genes were used for biolistic transformation and homologous integration was subsequently confirmed by PCR and Southern blot analysis. These results demonstrate that the emerging genomic resources, combined with in vitro transposition into plasmid DNAs from shotgun sequencing libraries or cloned PCR products, will facilitate high-throughput genetic analysis in Cryptococcus species.