Almeida A J, Carmona J A, Cunha C, Carvalho A, Rappleye C A, Goldman W E, Hooykaas P J, Leão C, Ludovico P, Rodrigues F
Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal.
Fungal Genet Biol. 2007 Dec;44(12):1387-98. doi: 10.1016/j.fgb.2007.04.004. Epub 2007 Apr 14.
We herein report the development of a molecular toolbox for the dimorphic fungus Paracoccidioides brasiliensis, specifically a more efficient transformation and a gene expression system. We evaluated several parameters that influence Agrobacterium tumefaciens-mediated transformation (ATMT), such as co-cultivation conditions and host cell susceptibility. Our results show that cellular recovery and air drying of A. tumefaciens:P. brasiliensis mixtures are essential for ATMT. Overall, our data indicate a transformation efficiency of 78+/-9 transformants/co-cultivation (5+/-1 transformants/10(6) target cells). P. brasiliensis GFP-expressing isolates were also constructed by insertion of the GFP gene under the control of several fungal promoters. RT-PCR, epifluorescence microscopy and flow cytometry analysis revealed Gfp visualization for all studied promoters but without significant differences in fluorescence and gene expression levels. Moreover, we present evidence for the occurrence of random single gene copy integration per haploid nuclei and the generation of homokaryon progeny, relevant for the future use in targeted mutagenesis and linking mutations to phenotypes.
我们在此报告了针对巴西副球孢子菌这种双态真菌开发的一个分子工具箱，具体而言是一种更高效的转化和基因表达系统。我们评估了影响根癌农杆菌介导转化（ATMT）的几个参数，如共培养条件和宿主细胞敏感性。我们的结果表明，根癌农杆菌与巴西副球孢子菌混合物的细胞复苏和空气干燥对ATMT至关重要。总体而言，我们的数据表明转化效率为78±9个转化体/共培养（5±1个转化体/10⁶个靶细胞）。通过在几个真菌启动子的控制下插入绿色荧光蛋白（GFP）基因，还构建了表达GFP的巴西副球孢子菌分离株。逆转录聚合酶链反应（RT-PCR）、落射荧光显微镜和流式细胞术分析显示，所有研究的启动子均能观察到绿色荧光蛋白（Gfp），但荧光和基因表达水平无显著差异。此外，我们提供了证据，证明每个单倍体细胞核发生随机单基因拷贝整合以及产生同核体后代，这对于未来在定向诱变以及将突变与表型联系起来的应用具有重要意义。
