新型隐球酵母cap70荚膜缺陷株转化系统的建立

Construction of Cryptococcus neoformans cap 70 transformation system

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。

Objective To construct a transformation system of Cryptococcus neoformans capsule deficient strain cap70. Methods (1)Obtained ura 5 mutants by screening the Cryptococcus neoformans cap70 using 5 fluoroorotic acid counter selection method, analyzed the obtaining strain by Southern blot and PCR; (2)Through the plasmid pCXJ18 and pCXJU which contained ura 5 gene from Cryptococcus neoformans and ura 3 from Kluyveromyces fragilils.as a selective marker, the ura 5 strains were transformed by electrotransformation and chemical transformation. Results Obtained ura 5 mutants, they were detected as lack of ura fuction. The chemical transformation method was first be used in Cryptococcus neoformans transformation. Conclusion A transformation system of Cryptococcus neoformans cap 70 has been established, which provides not only the basis for gene cloning from the species for studying gene function and gene expression.