【摘要】 目的:克隆烟曲霉sho1基因,并构建烟曲霉sho1基因突变株,了解该基因在烟曲霉应对环境变化中的作用。方法:通过基因同源性比对,在烟曲霉基因组中找出与酿酒酵母sho1基因同源的基因,PCR扩增烟曲霉sho1基因及其上、下游各约1.0kb的DNA片段,并将其重组到质粒pDHt/SK2中,再利用pyrG作为筛选标记构建重组质粒。将重组质粒转化烟曲霉AF293.1得到烟曲霉△sho1。观察烟曲霉△sho1和烟曲霉AF293(野生株,wt)在MM、CM、BHI这3种培养基上的生长速度,测定烟曲霉△sho1在含有氯化钠、丙三醇这两种渗透压物质培养基上的生长情况,并比较烟曲霉△sho1和wt对常用抗真菌药物的敏感性。结果:烟曲霉△sho1在CM、MM、BHI这3种培养基上的生长速度均慢于wt;△sho1在含渗透压物质小于1mol/L的培养基上菌落直径没有明显变化,在含渗透压物质大于1mol/L的培养基上菌落直径明显变小;烟曲霉△sho1和wt对抗真菌药物的敏感性没有差异。结论:sho1基因影响烟曲霉的生长速度,与培养基类型没有明显关系。烟曲霉sho1基因介导渗透压传导。sho1基因对烟曲霉的抗真菌药物敏感性没有影响。更多还原

【Abstract】 Objective:To construct the sho1 gene mutant of Aspergillus fumigatus,and to investigate the function of sho1 in adaptation to environmental changes.Methods:Saccharomyces cerivisiae sho1 gene homolog was identified by tblastn searches in A.fumigatus genome database.A PCR fragment,composed of the 5’ flanking sequence(approximately 1 kb) of sho1,sho1 ORF,and its 3’ flanking sequence(approximately 1 kb),was subcloned into a vector pDHt/SK2 with ApaⅠ/Xba Ⅰ enzyme to produce a recombinant plasmid PA.The selected marker pyrG cut from pLAX223 with Nco Ⅰ/Pst Ⅰ was cloned into PA,and PB was produced.The plasmid PB was transformed into competent Agrobacterium tumefaciens EHA105 by using the freeze/thaw method.The strain of A.tumefaciens was designated as At sho1.Then sho1 was produced via ATMT.The radial growth was observed and compared between sho1 and wild type(wt) on MM,CM and BHI medium,and the sensitivity of sho1 and wt to amphotericin B,itraconazole,voriconazole,and caspofungin investigated.Results:Radial growth had visible difference between sho1 and wt on MM,CM and BHI medium.sho1 mutant was sensitive to high osmotic pressure but not to low osmotic pressure.The susceptibility to antifungal drugs was similar between the sho1 and wt.Conclusion:Slow growth of the sho1 mutant has nothing to do with the different medium.The sho1 gene plays a role in response to osmotic pressure in A.fumigatus,but sho1 has a minor role in the susceptibility to the antifungal drugs despite the slow growth.更多还原