目的 构建烟曲霉额外拷贝菌株,了解额外拷贝烟曲霉sho1、pbs2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sho1、pbs2基因的额外拷贝菌株,采用Real-time PCR方法检测额外拷贝株中sho1、pbs2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sho1、pbs2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sho1、pbs2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△sho1、△pbs2对NaCl(1 mol/L)、H2O2(5mmol/L)、碱性pH (10.0)的敏感性高于野生株AF293.△sho1对刚果红(400 mg/L)的敏感性高于野生株,△pbs2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sho1或pbs2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.%Objective To explore the role of extra copies oisho 1 01 pbs 2 in resistance to hyperosmotic stress,oxidative stress, cell wall stress and alkaline pH stress in A. fumigatus . Methods The reconstructed plasmid harboring a sho 1 gene or a pbs 2 gene were transformed into A. fumigatus by using protoplast transformation method and the resulting strains were designated MCshol or MCpbs2. RT-PCR was used to detect the expression levels of sho 1 in MGshol and pbs 2 in MCpbs2 strains- Then strains harboring extra copies as well as sho 1 and pbs 2 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 sho 1 or pbs 2 showed higher expression levels of sho 1 or pbs 2 respectively. Extra copies of sho 1 or pbs 2 conferred resistance to NaCl (1 mol/L) , H2O2(5 mmol/L) , Congo red (400 mg/L)or alkaline pH (10.0) stress, sho 1 and pbs 2 deleted mutants showed higher sensitivity to NaCl ( 1 mol/L) ,H2G2(5 mmol/ L)and alkaline pH (10. 0) stress than the wild strain. A pbs 2 deleted mutant showed similar sensitivity to Congo red (400mg/L) compared to the wild strain. Conclusion Extra copies of sho 1 or pbs 2 conferred resistance to hyperosmotic stress, oxidative stress, cell wall stress and alkaline pH stress m A. fumigatus .
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