Agrobacterium-mediated insertional mutagenesis in Histoplasma capsulatum.
Agrobacterium-mediated insertional mutagenesis in Histoplasma capsulatum.
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DOI：
10.1007/978-1-61779-539-8_4
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发表时间：
2012
影响因子：
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通讯作者：
Olga Zemska;C. Rappleye
中科院分区：
文献类型：
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作者：
Olga Zemska;C. Rappleye
全基因组诱变是一种用于鉴定有助于感兴趣表型的新基因的有力方法。根癌农杆菌介导的遗传转化（ATMT）是一种有效的插入诱变剂。在荚膜组织胞浆菌中，由农杆菌转移的T-DNA元件稳定地整合到基因组中，并且大多数突变体含有插入序列的单拷贝。T-DNA序列有助于通过半特异性PCR技术、质粒拯救或反向PCR确定插入侧翼的基因组序列。我们提出了优化的程序产生插入突变体inH。农杆菌介导的转化，并将其用于正向和反向遗传方法。
Genome-wide mutagenesis is a powerful method for identifying new genes that contribute to a phenotype of interest. For many fungal pathogens of plants and animals,Agrobacterium tumefaciens-mediated transformation (ATMT) serves as an efficient insertional mutagen. InHistoplasma capsulatum, the T-DNA element transferred byAgrobacteriumstably integrates into the genome, and the majority of mutants contain single copies of the inserted sequence. The T-DNA sequence facilitates the determination of the genomic sequence flanking the insertion through hemi-specific PCR techniques, plasmid rescue, or inverse PCR. We present optimized procedures for generating insertional mutants inH. capsulatumusingAgrobacterium-mediated transformation and using this for forward and reverse genetic approaches.