Casado-Del Castillo Virginia, MacCabe Andrew P, Orejas Margarita
Instituto de Agroquímica y Tecnología de Alimentos (IATA), Consejo Superior de Investigaciones Científicas (CSIC), c/Catedrático Agustín Escardino Benlloch 7, 46980 Paterna, Valencia, Spain.
J Fungi (Basel). 2021 Nov 12;7(11):961. doi: 10.3390/jof7110961.
Protoplast transformation for the introduction of recombinant DNA into is technically demanding and dependant on the availability and batch variability of commercial enzyme preparations. Given the success of -mediated transformation (ATMT) in diverse pathogenic fungi, we have adapted this method to facilitate transformation of . Using suitably engineered binary vectors, gene-targeted ATMT of non-homologous end-joining (NHEJ) mutant conidia has been carried out for the first time by complementation of a nutritional requirement (uridine/uracil auxotrophy). Site-specific integration in the Δ host genome occurred at high efficiency. Unlike other transformation techniques, however, cross-feeding of certain nutritional requirements from the bacterium to the fungus was found to occur, thus limiting the choice of auxotrophies available for ATMT. In complementation tests and also for comparative purposes, integration of recombinant cassettes at a specific locus could provide a means to reduce the influence of position effects (chromatin structure) on transgene expression. In this regard, targeted disruption of the locus permitted visual identification of transformants carrying site-specific integration events by conidial colour (white), even when auxotrophy selection was compromised due to cross-feeding. The protocol described offers an attractive alternative to the protoplast procedure for obtaining locus-targeted transformants.
将重组DNA导入原生质体的转化技术要求较高，且依赖于商业酶制剂的可用性和批次差异。鉴于农杆菌介导的转化（ATMT）在多种致病真菌中取得的成功，我们对该方法进行了改进，以促进[具体物种名称]的转化。使用经过适当工程改造的二元载体，通过补充营养需求（尿苷/尿嘧啶营养缺陷型），首次对[具体物种名称]非同源末端连接（NHEJ）突变分生孢子进行了基因靶向ATMT。在Δ宿主基因组中高效发生了位点特异性整合。然而，与其他转化技术不同的是，发现存在从细菌到真菌的某些营养需求的交叉喂养现象，从而限制了可用于ATMT的营养缺陷型的选择。在互补试验以及比较目的中，重组盒在特定位点的整合可以提供一种减少位置效应（染色质结构）对转基因表达影响的方法。在这方面，即使由于交叉喂养导致营养缺陷型选择受到影响，靶向破坏[具体基因名称]位点也允许通过分生孢子颜色（白色）直观鉴定携带位点特异性整合事件的转化体。所描述的方案为获得位点靶向的[具体物种名称]转化体提供了一种有吸引力的替代原生质体程序的方法。
