[Effect of sho1 gene of Aspergillus fumigatus on adaptation to osmotic pressure and on sensitivity of antifungal drugs].
[Effect of sho1 gene of Aspergillus fumigatus on adaptation to osmotic pressure and on sensitivity of antifungal drugs].
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发表时间：
2009-04
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通讯作者：
Yansen Ma;Jian Qiao;Wei Liu;Ruo-yu Li
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作者：
Yansen Ma;Jian Qiao;Wei Liu;Ruo-yu Li
目的构建烟曲霉sho 1基因突变株，研究sho 1基因在烟曲霉适应环境变化中的作用。方法通过对酿酒酵母sho 1基因同源序列的分析，在A.烟曲霉基因组数据库。将sho 1的5'侧翼序列（约1 kb）、sho 1 ORF及其3'侧翼序列（约1 kb）组成的PCR片段亚克隆到含有Apa I/Xba I酶的载体pDHt/SK 2中，以产生重组质粒PA。用NcoI/Pst I从pLAX 223上切下选择性标记pyrG，将其克隆到PA中，并产生PB。用冻融法将质粒PB转化到感受态根癌农杆菌EHA 105中。A. tumefaciens命名为At sho 1。然后通过ATMT产生三角形upsho 1。观察并比较了triangle up sho 1和野生型（wt）在MM、CM和BHI培养基上的放射状生长，并研究了triangle up sho 1和wt对阿替霉素B、伊曲康唑、伏立康唑和卡泊芬净的敏感性。结果在MM、CM和BHI培养基上，三角形上sho 1和野生型之间的径向生长有明显差异。突变体sho 1对高渗透压敏感，对低渗透压不敏感。三角形上sho 1和野生型对抗真菌药物的敏感性相似。结论突变株生长缓慢与培养基的不同无关。sho 1基因在A.尽管生长缓慢，但SHO 1在抗真菌药物的敏感性中具有较小的作用。
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/XbaI enzyme to produce a recombinant plasmid PA. The selected marker pyrG cut from pLAX223 with NcoI/PstI 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 triangle upsho1 was produced via ATMT. The radial growth was observed and compared between triangle up sho1 and wild type (wt) on MM, CM and BHI medium, and the sensitivity of triangle up sho1 and wt to amphotericin B, itraconazole, voriconazole, and caspofungin investigated. RESULTS Radial growth had visible difference between triangle up sho1 and wt on MM, CM and BHI medium. triangle up sho1 mutant was sensitive to high osmotic pressure but not to low osmotic pressure. The susceptibility to antifungal drugs was similar between the triangle up sho1 and wt. CONCLUSION Slow growth of the triangle up 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.