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目的 :克隆烟曲霉角鲨烯环氧化酶基因并探讨其与特比萘芬抗烟曲霉活性的关系。方法 :利用聚乙二醇-甘油法将 0 .5 μg烟曲霉基因组DNA文库转化pyrG 烟曲霉AF2 93.1原生质体 ;在含有特比萘芬的最低培养基上筛选耐特比萘芬的pyrG +转化子 ,然后对介导特比萘芬耐药的烟曲霉基因进行序列分析并进行基因克隆 ;最后将克隆的基因重新转化到烟曲霉中以确认其与特比萘芬敏感性之间的关系。结果 :从 5× 10 4个转化子中得到一个耐特比萘芬的pyrG +转化子 ,该转化子表现出特比萘芬特异性耐药性且与质粒高度相关 ;序列分析发现 ,是烟曲霉角鲨烯环氧化酶基因介导了特异性特比萘芬耐药 ;将该基因克隆后重新转化到烟曲霉中 ,结果敏感的烟曲霉再次获得特异性特比萘芬耐药性。结论 :首次获得烟曲霉角鲨烯环氧化酶基因 ,并发现额外拷贝的该基因可以导致特异性特比萘芬耐药这一新型机制。
OBJECTIVE: To clone the gene of squalene epoxidase from Aspergillus fumigatus and investigate its relationship with the activity of terbinafine against Aspergillus fumigatus. Methods: The genomic DNA library of 0.5 μg Aspergillus fumigatus genomic DNA was transformed into Aspergillus fumigatus AF2 93.1 protoplasts by polyethylene glycol-glycerol method. The pyrG + transformants of terbinafine were screened on the lowest medium containing terbinafine Then, the genes of Aspergillus fumigatus mediated by terbinafine were sequenced and cloned. Finally, the cloned gene was transformed into Aspergillus fumigatus to confirm its relationship with the sensitivity of terbinafine. Results: A 5-year-old pyrG + transformant resistant to terbinafine was obtained from 5 × 10 4 transformants. This transformant shows the specific resistance to terbinafine and is highly related to the plasmid. Sequencing analysis showed that the transformant was tobacco Aspergillus squalene epoxidase gene mediates specific terbinafine resistance; this gene was cloned and then transformed back to Aspergillus fumigatus, with the result that the sensitive A. fumigatus was again given specific terbinafine resistance. CONCLUSIONS: The first Aspergillus fumigatus squalene epoxidase gene was obtained and an additional copy of this gene was found to result in a novel mechanism of specific terbinafine resistance.