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【目的】新型隐球酵母(Cryptococcus neoformans)是人类重要致病真菌,主要毒性因子之一漆酶的表达受葡糖糖阻遏,机制未知。本文拟寻找参与葡萄糖阻遏的关键基因。【方法】建立根癌农杆菌介导的转化方法(Agrobacterium tumefanciens-mediated transformation,ATMT)建立一个容量约200000的随即插入突变文库,在高浓度葡萄糖条件下从中筛选葡萄糖去阻遏的突变株。通过Southern确定突变株中T-DNA的拷贝数,利用反向PCR获得依赖葡萄糖的漆酶阻遏基因序列。【结果】筛选到了30株葡萄糖去阻遏突变株,Southern blot发现83%的葡萄糖去抑制突变株含有单个T-DNA拷贝。初步鉴定了可能参与漆酶阻遏的10个不同生物学功能基因,如参与碳水化合物的代谢,固醇的合成,几丁质的合成,GPI脂锚钩的合成等等。【结论】ATMT突变策略可以找到一些参与漆酶葡萄糖阻遏的关键基因,为理解漆酶在致病过程中的作用机制和工业改进漆酶活性提供参考。
【Objective】 Cryptococcus neoformans is an important pathogenic fungus in humans. The expression of laccase, one of the major toxic factors, is repressed by glucose and its mechanism is unknown. This article is intended to find the key genes involved in glucose repression. 【Method】 Agrobacterium tumefanciens-mediated transformation (ATMT) was used to establish a library of about 200000 inserted mutants. The mutants with de-repression of glucose were screened under high glucose concentration. The copy number of T-DNA in the mutant strain was determined by Southern blotting, and the glucose-dependent laccase repressor gene sequence was obtained by reverse PCR. 【Result】 Thirty glucose-deprivation mutants were screened. Southern blot showed that 83% of glucose-depressant mutants contained a single copy of T-DNA. Preliminary identification of 10 different biological function genes that may be involved in laccase repression, such as carbohydrate metabolism, sterol synthesis, chitin synthesis, GPI lipid anchor synthesis and so on. 【Conclusion】 The ATMT mutation strategy can find some key genes that participate in the repression of laccase glucose, which may provide a reference for understanding the mechanism of action of laccase in pathogenesis and industrial improvement of laccase activity.