新生隐球菌CAP64荚膜缺陷株ura5突变株的筛选和鉴定  

Screening and identification of ura5 mutants of Cryptoccocus neoformans capsule-deficient strain CAP64

作　　者：郭秀军[1] 廖万清[1] 任大明[2] 王荫榆[2] 霍克克[2] 

机构地区：[1]第二军医大学长征医院皮肤科,上海200003 [2]复旦大学遗传研究所微生物组,上海200433

出　　处：《第二军医大学学报》2004年第4期418-421,共4页Academic Journal of Second Military Medical University

基　　金：国家自然科学基金 ( 3 95 70 0 43 )

摘　　要：目的 :筛选和鉴定新生隐球菌 CAP6 4荚膜缺陷株 ura5突变株。方法 :(1 )用体外基因重组技术将 G4 1 8抗性基因插入到新生隐球菌 1 .2 kb的 ura5基因中 ;(2 )用电转化方法将体外重组片段导入受体菌 ;(3)利用 5 -氟乳清酸 (5 - FOA)反筛选法筛选 ura5突变株 ;(4 )用遗传学鉴定营养缺陷株的方法鉴定突变体。结果 :1株突变株 ura5基因的序列与重组片段相同 ;其Southern杂交显示在 2 0 kb和 7kb处出现 2条杂交条带 ;该突变株能在 SDU、SDU和 YEPD/ G4 1 8平板上生长 ,不能在 SD平板生长 ;其生长曲线显示突变株生长速度依赖于尿嘧啶的提供量。 结论 :获得了 1株新生隐球菌 CAP6 4荚膜缺陷株 ura5突变株 ,建立了新生隐球菌 CAP6 4荚膜缺陷株转化系统 ,为 CAP6Objective:To screen and identify ura5 mutants of Cryptoccocus neoformans capsule deficient strains CAP64. Methods:(1)G418 resistance gene was inserted into 1.2 kb ura5 gene of Cryptoccocus neoformans by gene recombination in vitro ;(2)Genomic ura5 gene was displaced by the recombined gene with eletroporation method;(3)5 FOA was used to screen for ura5 mutants;(4)The ura5 mutants were identified by genetics methods.Results:Sequence of one ura5 mutant was the same as recombination fragment.Southern analysis of mutants showed that there were 2 hybridization bands(20 kb and 7 kb).Mutants were able to grow on SDU and YEPD/G418 medium,but not on SD medium.The growth chart showed that growth speed of mutants was depended on the supply of uracil. Conclusion:A uracil defecient mutant has been abtained,and a transformation system of Cryptoccocus neoformans capsule deficient strain CAP64 has been established,which provides a basis for further study of CAP64 gene function.

关 键 词：新生隐球菌 CAP64 荚膜缺陷株 ura5 突变株 筛选 鉴定 

分 类 号：R379.5[医药卫生—病原生物学]