State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Science and Technology, Guangxi University, 100 Daxue Road, Nanning, Guangxi, 530004, PR China.
Fungal Biol. 2017 Jun-Jul;121(6-7):615-623. doi: 10.1016/j.funbio.2017.04.005. Epub 2017 May 1.
To improve the gene targeting frequency (GTF) in the lignocellulolytic filamentous fungus Penicillium oxalicum HP7-1, the non-homologous end-joining (NHEJ) gene ligD was deleted. The obtained PoligD deletion mutant ΔPoligD showed no apparent defect in cellulase production, growth rate, and sensitivity towards osmotic stress and mutagen ethyl methanesulphonate (EMS), while increased sensitivity to high concentrations of methyl methanesulfonate (MMS). Deletion of PoligD gene resulted in significantly increased GTFs at three different loci in P. oxalicum, which are even higher than those in Poku70 deletion mutant. The GTF in ΔPoligD at PoargB (reached 97 %) and PoagaA (reached 90 %) loci increased 5.1- and 1.2-fold compared with that in wild-type strain (WT), while at the Podpp4 locus GTF was up to 27 % in ΔPoligD but close to 0 % in WT, with 0.5 kb homologous flanking regions. Furthermore, the argB and agaA nutritional selection in P. oxalicum was demonstrated and the PoargB and PoagaA genes could be used as selective markers in this fungus. Thus, the PoligD deletion mutant can be an important tool for the functional analysis of genes in P. oxalicum.
为了提高木质纤维素分解丝状真菌草酸青霉 HP7-1 的基因打靶频率（GTF），删除了非同源末端连接（NHEJ）基因 ligD。所得的 PoligD 缺失突变体ΔPoligD 在纤维素酶生产、生长速率以及对渗透压胁迫和诱变剂甲基甲磺酸乙酯（EMS）的敏感性方面没有明显缺陷，而对高浓度甲基甲磺酸甲酯（MMS）的敏感性增加。PoligD 基因的缺失导致草酸青霉中的三个不同基因座的 GTF 显著增加，甚至高于 Poku70 缺失突变体。ΔPoligD 在 PoargB（达到 97%）和 PoagaA（达到 90%）基因座的 GTF 分别比野生型菌株（WT）增加了 5.1 倍和 1.2 倍，而在 Podpp4 基因座的 GTF 在ΔPoligD 中高达 27%，而在 WT 中几乎为 0%，侧翼有 0.5kb 的同源区域。此外，还在草酸青霉中进行了 argB 和 agaA 营养选择，并证明了 PoargB 和 PoagaA 基因可作为该真菌中的选择性标记。因此，PoligD 缺失突变体可以成为草酸青霉中基因功能分析的重要工具。
