Identification and characterization of an anti-oxidative stress-associated mutant of Aspergillus fumigatus transformed by Agrobacterium tumefaciens.
Identification and characterization of an anti-oxidative stress-associated mutant of Aspergillus fumigatus transformed by Agrobacterium tumefaciens.
复制标题
根癌农杆菌转化的烟曲霉抗氧化应激相关突变体的鉴定和表征。
DOI：
10.3892/mmr.2016.4839
复制
发表时间：
2016-03
影响因子：
3.4
通讯作者：
Wang L
中科院分区：
文献类型：
--
作者：
Fan Z;Yu H;Guo Q;He D;Xue B;Xie X;Yokoyama K;Wang L
烟曲霉是一种最常见的条件致病真菌，能在多种环境条件下生存。真菌氧化还原动态平衡的维持有赖于免疫细胞或自身细胞器产生的活性氧物种与激活的抗氧化应激机制之间的有机调节。为了研究这一机制，本研究获得了一些由农杆菌介导的随机插入的烟曲霉突变体。此外，还建立了高通量过氧化氢筛选系统，用于检测约1000个突变株。共有100个突变体表现出过氧化氢敏感性的变化，其中AFM2658突变体的敏感度显著增加。对该突变体进行了进一步的研究，利用热不对称交错聚合酶链式反应对该突变体的序列进行了鉴定。这表明插入位点位于第2染色体afu1_92上，并敲除了96bp的序列，该序列部分由位于完整膜蛋白编码区和螺旋-环-螺旋转录因子编码区之间的序列组成。观察到与抗氧化应激相关的mRNAs水平下降，并用荧光检测到活性氧物种的增加。本研究的结果表明，该序列可能在氧化胁迫下对烟曲霉菌具有保护作用。
Aspergillus fumigatus is one of the most common opportunistic pathogenic fungi, surviving in various environmental conditions. Maintenance of the redox homeostasis of the fungus relies upon the well-organized regulation between reactive oxygen species generated by immune cells or its own organelles, and the activated anti-oxidative stress mechanism. To investigate such a mechanism, the present study obtained a number of randomly-inserted mutants of A. fumigatus, mediated by Agrobacterium tumefaciens. In addition, a high throughput hydrogen peroxide screening system was established to examine ~1,000 mutants. A total of 100 mutants exhibited changes in hydrogen peroxide sensitivity, among which a significant increase in sensitivity was observed in the AFM2658 mutant. Further investigations of the mutant were also performed, in which the sequence of this mutant was characterized using thermal asymmetric interlaced-polymerase chain reaction. This revealed that the insertion site was located on chromosome 2 afu1_92, and the 96 bp sequence was knocked out, which partially comprised a sequence localized between the integral membrane protein coding region and the helix-loop-helix transcription factor coding region. A decrease in the levels of anti-oxidative stress-associated mRNAs were observed, and an increase in reactive oxygen species were detected using fluorescence. The results of the present study demonstrated that this sequence may have a protective role in A. fumigatus in the presence of oxidative stress.