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目的 构建新型隐球菌荚膜缺陷株cap70的转化系统。方法 (1)在新型隐球菌的荚膜缺陷株cap70中利用 5 氟乳清酸 (5 FOA)反选择法筛选尿嘧啶合成基因突变株 (cap70ura5 ) ,并用Southern杂交和PCR方法对突变株作进一步验证 ;(2 )利用以ura5及ura3为选择标记的质粒载体pCXJ18及pCXJU ,用电转化法和化学转化法转化ura5突变株。结果 筛选到ura5突变株 ,经验证该突变株尿嘧啶合成功能缺失 ;首次用化学转化法在新型隐球菌的转化中获得高效率转化。结论 首次建立了新型隐球菌cap70荚膜缺陷株的转化系统 ,为克隆与荚膜形成有关的新基因提供了基础 ,并为以基因中断、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 new type of capsular transformation system of Cryptococcus capsulatus defect. Methods (1) The uracil synthetic gene mutant (cap705u5) was screened by 5 FOA reverse selection method in the capsular defect of Capnococcus neoformans, and the mutant was further characterized by Southern blot and PCR (2) Ura5 mutant was transformed by electrotransformation and chemical transformation using plasmid vector pCXJ18 and pCXJU with ura5 and ura3 as selectable markers. Results The ura5 mutant was screened and the uracil synthesis function was absent. The first transformation of Cryptococcus neoformans with chemical transformation was efficient. Conclusions The transformation system of the capsular defect of Cryptococcus neoformans cap70 was established for the first time, which provided a basis for cloning new genes related to capsule formation. It was also for the purpose of gene disruption and gene replacement to study gene function and gene expression Conditions