新生隐球菌cap60荚膜缺陷株转化系统的构建

Construction of the Cryptococcus Neoformans cap60 Transformation System

目的 构建新生隐球菌荚膜缺陷株cap60的转化系统。方法与结果①在新生隐球菌的荚膜缺陷株cap60中利用 5 -氟乳清酸 ( 5 FOA)反选择法筛选到一个尿嘧啶合成基因突变株 (cap60ura5 ) ;②利用带有以新生隐球菌ura5基因为选择标记的质粒载体 pCntel1 d通过电转化法和化学转化法成功地转化该ura5突变株 ;③经质粒稳定性检验和Southern杂交分析表明质粒载体pCntel1 d在新生隐球菌cap60ura5菌株中能以整合和游离两种状态存在。结论首次建立了新生隐球菌cap60荚膜缺陷株的转化系统 ,这不仅为克隆与荚膜形成有关的新基因提供了基础 ,还为以基因中断、基因替代方法研究基因功能及基因表达等方面的工作创造了条件。

Objective To construct a transformation system of Cryptococcus neoformans (C neoformans) capsule deficient strain cap60 Methods and Results The results included: (1) A ura 5 mutant was obtained by screening the C neoformans cap60 using 5 fluoroorotic acid counter selection method (2) By electrotransformation and chemical transformation, the ura5 strain was successfully transformed through the plasmid pCntel1 d which contained the ura5 gene from C neoformans as a selective marker (3) The plasmid stability test and Southern blot analysis indicated there were two status of plasmid pCntel1 d in C neoformans transformants: integrated or episomal plasmid Conclusion A transformation system of C neoformans cap60 has been established, which provides not only the basis for gene cloning from the species but also for studying gene function and gene expression