金属还原酶FreB2对烟曲霉铁吸收及氧化压力应答的影响

利用构建的烟曲霉金属还原酶基因(AFUA-1G00350,Fre B2)缺失突变株,对烟曲霉金属还原酶基因Fre B2功能进行初步研究,为揭示该基因与烟曲霉的致病关系提供依据。比较野生株和基因缺失突变株在AMM和无铁AMM液体培养基中生长时高铁还原酶的活性,绘制不同时间野生株和基因缺失突变株在AMM和无铁AMM液体培养基中生长时高铁还原酶活性曲线。利用Real-Time PCR方法分析Sre A、Sid A、Fet C、Ftr A和Fre B这些与铁的吸收相关基因的mRNA的表达量变化。测定野生株和基因缺失突变株对氧化压力的敏感性及胞内活性氧物质含量。不论在AMM液体培养基中还是在无铁AMM液体培养基中培养时,突变株高铁还原酶的活性都明显高于野生株高铁还原酶活性。与野生株相比培养60 h时,突变株Sre A、Sid A、Fet C、Ftr A和Fre B这些与铁的吸收相关基因的表达量出现明显上调。氧化压力敏感性实验显示,基因缺失突变株对H2O2的敏感性显著增强,同时胞内活性氧物质含量明显增多。金属还原酶基因Fre B2在烟曲霉铁吸收及氧化压力应答过程中发挥作用;烟曲霉与铁吸收相关基因之间存在功能互补效应。

The Effects of Metalloreductase FreB2 on the Uptake of Iron and Response to Oxidative Stress in Aspergillus fumigatus

The function of a metalloreductase gene FreB2 of Aspergillus fumigatus was initially studied by the establishment of metalloreductase gene (AFUA-1G00350, FreB2) deletion mutant strain of A. fumigatus, in order to explore the pathogenic relation of A. fumigatus and the gene to provide foundation. The metalloreductase activity of wild type strain and gene deletion strain of A. fumigatus were compared in AMM and iron free AMM medium liquid during their growth for the activity of metalloreductase, and draw the metalloreductase activity curve of the wild type strain and gene deletion strain in AMM and iron free AMM liquid medium during their growth. Using Real Time PCR to analyze the mRNA expression variation of SreA, SidA, FetC, FtrA, and FreB these related genes to iron assimilation was significantly up regulated. The sensitivity to oxidation stress experiment showed that the sensitivity of gene deletion mutant strain to H₂O₂ was significantly strengthened, at the same time the intracellular active oxygen substance significantly increased. Metalloreductase FreB2 played role during the process of iron assimilation and oxidation stress response of A. fumigatus; therefore, there existed a functionally mutual complementary effect on A. fumigatus and iron assimilation related genes.