Department of Pathogenobiology, Norman Bethune College of Medicine, Jilin University Mycology Research Center, Jilin University, Changchun 130021, China.
J Microbiol Methods. 2011 Mar;84(3):418-22. doi: 10.1016/j.mimet.2011.01.017. Epub 2011 Jan 21.
Sporothrix schenckii is a dimorphic pathogenic fungus that causes human and animal sporotrichosis globally. Here we developed and optimized an Agrobacterium tumefaciens-mediated transformation (ATMT) system of S. schenckii for insertional mutagenesis. The transformation efficiency reached more than 600 transformants per 10(6) conidia. Using this protocol enabled us to obtain a large number of T-DNA insertional mutants within a short experimental period. Several mutants with altered phenotypes were obtained during the transformation experiments. The mutants displayed mitotic stability. Transferred DNA (T-DNA) flanking sequences were cloned by thermal asymmetric interlaced PCR (TAIL-PCR). Our results demonstrated that the ATMT system can be an effective tool for insertional mutagenesis in S. schenckii. This is the first report of a suitable mutagenesis system which may provide valuable mutants and information for both forward and reverse genetics research in the future for this medically important fungus.
申克孢子丝菌是一种二态致病真菌，可引起全球人类和动物的孢子丝菌病。在此，我们开发并优化了申克孢子丝菌的农杆菌介导转化（ATMT）系统，用于插入诱变。转化效率达到每 10^6 个分生孢子超过 600 个转化体。使用该方案，我们能够在短时间内获得大量 T-DNA 插入突变体。在转化实验中获得了几个表型改变的突变体。突变体表现出有丝分裂稳定性。通过热不对称交错 PCR（TAIL-PCR）克隆了转移 DNA（T-DNA）侧翼序列。我们的结果表明，ATMT 系统可以成为申克孢子丝菌插入诱变的有效工具。这是首次报道适合该医学上重要真菌的正向和反向遗传学研究的诱变系统，它可能为未来提供有价值的突变体和信息。
