利用根癌农杆菌T-DNA 插入突变寻找参与漆酶葡萄糖阻遏的关键基因

【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。

Agrobacterium tumefaciens-mediated transformation (ATMT) for the screening for genes involved in laccase glucose repression in the pathogenic yeast Cryptococcus neoformans

Objective] To identify genes in glucose repression of laccase in the human pathogen Cryptococcus neoformans.Methods] We created a random insertional mutagenesis library containing over 200000 transformants by Agrobacterium tumefaciens-mediated transformation(ATMT).We screened the glucose derepression mutants under high-glucose condition and obtained the genes for glucose repression of laccase via inverse polymerase chains reaction(PCR).Results] Totally,we isolted 30 glucose derepression mutants from the library.We found that that 83% of the mutants contain a single T-DNA via Southern blot.We preliminarily identified 10 genes,which fall into a broad range of biological processes including: carbohydrate metabolism,sterol biosynthesis,chitin biosynthesis and glycosylphatidylinositol(GPI) anchor biosynthesis.Additionally,we found that three glucose derepression mutants have a single T-DNA insertion in the promoter region of LAC1,which encodes cryptococcal laccase.Conclusion] As an effective way,ATMT can be utilized for identifying genes in glucose repression of laccase,which sheds lights on the roles of laccase in virulence and provides information for laccase production in industry.