新生隐球菌COP9复合体基因CSN6的敲除与鉴定  

Construction of knockout strains of The COP9 signalosome subunit 6(CSN6)in Cryptococcus neoformans

作　　者：杨雅骊[1,2] 张超[1,2] 孟云芳[1,2] 法振宗 周兆婧 赵静宇[1,2] 方伟[1,2] 廖万清[1,2] 

机构地区：[1]上海长征医院皮肤病与真菌病研究所全军真菌病重点实验室 [2]第二军医大学附属长征医院皮肤科,上海200003

出　　处：《中国真菌学杂志》2014年第6期321-324,330,共5页Chinese Journal of Mycology

基　　金：国家973项目(2013CB531601);上海市自然科学基金(12ZR1454400;12JC1411000)

摘　　要：目的构建新生隐球菌COP9复合体蛋白元件Csn6的基因同源重组敲除框,并通过基因枪转化系统敲除CSN6基因。方法应用生物信息学方法获得COP9复合体蛋白元件的基因信息,采用套叠PCR的方法,构建包含报告基因NEO和CSN6基因ORF两侧上下游同源DNA片段的同源重组框。应用基因枪将其转化入新生隐球菌感受态细胞,通过PCR和DNA测序对遗传霉素(G418)耐受的阳性克隆子进行筛选与验证。结果成功构建了新生隐球菌基因突变株csn6裣。结论 COP9复合体亚基CSN6基因突变株的构建,为今后新生隐球菌COP9复合体的分子致病机制研究奠定基础。Objective To construct the homologous recombination cassette of COP9/signalosome component gene CSN 6 and generate its deletion mutant via biolistic transformation. Methods We first got the DNA sequence of COP9/signalsome components by bioinformatics analysis. On the basis, we constructed the homologous recombination cassette of CSN6 via o- verlap PCR and transformed them into Cryptococcal competent cells via Particle Delivery System. The positive transformants were screened via YPD argar including geneticin （G418）, which were then identified via diagnostic PCR and DNA sequen- cing. Results CSN6 deletion strains were successfully constructed. Conclusions This study laid the groundwork for follow- ing functional studies of COP9/signalosome in Cryptococcus neoformans.

关 键 词：新生隐球菌 COP9信号复合体 CSN6 致病机制 

分 类 号：R379.5[医药卫生—病原生物学]