烟曲霉sho1、pbs2基因额外拷贝对几种应激能力的影响

目的构建烟曲霉额外拷贝菌株,了解额外拷贝烟曲霉sho 1、pbs 2基因能否增强菌株对高渗透压、过氧化氢(H2O2)、碱性pH、刚果红应激的抵抗能力,探讨HOG通路(high osmolarity glycerol pathway)参与的应激反应。方法用原生质体法构建分别含有烟曲霉sho 1、pbs 2基因的额外拷贝菌株,采用Real-time PCR方法检测额外拷贝株中sho 1、pbs 2的表达情况。观察并比较缺陷株、额外拷贝株对NaCl(1 mol/L)、H2O2(5 mmol/L)、刚果红(400 mg/L)及碱性pH(10.0)应激的反应。结果获得了含有烟曲霉sho 1、pbs 2基因的额外拷贝菌株MCsho1、MCpbs2,和含空白质粒的对照株Empty。额外拷贝株sho 1、pbs 2的表达水平增高,对NaCl(1 mol/L)、H2O2(5 mmol/L)、刚果红(400 mg/L)、碱性pH(10.0)应激的抵抗强于Empty。MCpbs2对这些应激的抵抗较MCsho1更显著。烟曲霉缺陷株△sho 1、△pbs 2对NaCl(1 mol/L)、H2O2(5mmol/L)、碱性pH(10.0)的敏感性高于野生株AF293。△sho 1对刚果红(400 mg/L)的敏感性高于野生株,△pbs 2对刚果红的敏感性与野生株比,无显著差别。结论额外拷贝烟曲霉sho 1或pbs 2基因能增强菌株对高渗透压、氧化压力、刚果红、碱性pH应激的抵抗能力。

The effect of extra copies of sho1 or pbs2 gene on the adaptive ability of Aspergillus fumigatus to several stresses

Objective To explore the role of extra copies of sho1 or pbs2 in resistance to hyperosmotic stress,oxidative stress,cell wall stress and alkaline pH stress in A.fumigatus.Methods The reconstructed plasmid harboring a sho1 gene or a pbs2 gene were transformed into A.fumigatus by using protoplast transformation method and the resulting strains were designated MCsho1 or MCpbs2.RT-PCR was used to detect the expression levels of sho1 in MCsho1 and pbs2 in MCpbs2 strains.Then strains harboring extra copies as well as sho1 and pbs2 deleted mutants were treated with NaCl,H2O2,Congo red and alkaline pH stresses and the resistance phenotypes were observed.Results Mutants harboring extra copies of sho1 or pbs2 showed higher expression levels of sho1 or pbs2 respectively.Extra copies of sho1 or pbs2 conferred resistance to NaCl(1 mol/L),H2O2(5 mmol/L),Congo red(400 mg/L)or alkaline pH(10.0)stress.sho1 and pbs2 deleted mutants showed higher sensitivity to NaCl(1 mol/L),H2O2(5 mmol/L)and alkaline pH(10.0)stress than the wild strain.△pbs2 deleted mutant showed similar sensitivity to Congo red(400mg/L) compared to the wild strain.Conclusion Extra copies of sho1 or pbs2 conferred resistance to hyperosmotic stress,oxidative stress,cell wall stress and alkaline pH stress in A.fumigatus.