Department of Genetics, The University of Melbourne, Parkville, Victoria 3010, Australia.
Fungal Genet Biol. 2012 Oct;49(10):772-8. doi: 10.1016/j.fgb.2012.08.003. Epub 2012 Aug 17.
Penicillium marneffei is an opportunistic pathogen of humans and displays a temperature dependent dimorphic transition. Like many fungi, exogenous DNA introduced by DNA mediated transformation is integrated randomly into the genome resulting in inefficient gene deletion and position-specific effects. To enhance successful gene targeting, the consequences of perturbing components of the non-homologous end joining recombination pathway have been examined. The deletion of the KU70 and LIG4 orthologs, pkuA and ligD, respectively, dramatically enhanced the observed homologous recombination frequency leading to efficient gene deletion. While ΔpkuA was associated with reduced genetic stability over-time, ΔligD represents a suitable recipient strain for downstream applications and combined with a modified Gateway™ system for the rapid generation of gene deletion constructs, this represents an efficient pipeline for characterizing gene function in P. marneffei.
马尔尼菲青霉是一种人类机会致病菌，具有温度依赖性的二态性转变。像许多真菌一样，通过 DNA 介导的转化引入的外源 DNA 随机整合到基因组中，导致基因缺失效率低下和位置特异性效应。为了提高基因靶向的成功率，研究了干扰非同源末端连接重组途径的成分的后果。分别删除 KU70 和 LIG4 的同源物 pkuA 和 ligD，显著提高了观察到的同源重组频率，从而实现了有效的基因缺失。虽然 ΔpkuA 与随时间推移遗传稳定性降低有关，但 ΔligD 代表了下游应用的合适受体菌株，并且与改良的 Gateway™系统结合用于快速生成基因缺失构建体，这代表了在马尔尼菲青霉中表征基因功能的有效途径。
