Plourde Karine V, Jacobi Volker, Bernier Louis
Centre d'Etude de la Forêt, Faculté de Foresterie et de Géomatique, Université Laval, Québec, QCG1V0A6, Canada.
Can J Microbiol. 2008 Sep;54(9):797-802. doi: 10.1139/w08-068.
We used insertional mutagenesis to produce genetically tagged mutants of the Dutch elm disease fungus Ophiostoma novo-ulmi subsp. novo-ulmi. We first optimized transformation of O. novo-ulmi protoplasts by the restriction enzyme mediated integration method. A concentration of 80 U of HindIII with 108 fungal protoplasts and 5 microg of plasmid DNA was the most efficient for generating a high number of O. novo-ulmi mutants carrying a single insertion in their genome. Mycelium- and yeast-like growth kinetics of 24 O. novo-ulmi mutants were evaluated in vitro. Flanking sequences were successfully recovered in 8% of the transformants analyzed. Some mutant phenotypes appeared to result from gene disruption events, whereas others likely involved modifications of noncoding regions. Several nuclear loci that control vegetative growth and could potentially impact parasitic fitness were successfully tagged.
我们利用插入诱变技术来产生荷兰榆树病真菌新榆长喙壳菌新榆亚种的基因标记突变体。我们首先通过限制酶介导整合方法优化了新榆长喙壳菌原生质体的转化。80单位的 HindIII与10⁸个真菌原生质体以及5微克质粒DNA的浓度对于产生大量在其基因组中携带单个插入的新榆长喙壳菌突变体最为有效。在体外评估了24个新榆长喙壳菌突变体的菌丝体和酵母样生长动力学。在所分析的转化体中，8%成功回收了侧翼序列。一些突变体表型似乎是由基因破坏事件导致的，而其他表型可能涉及非编码区的修饰。成功标记了几个控制营养生长并可能影响寄生适合度的核基因座。
