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

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

【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] 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 isohed 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.