新生隐球菌COP9复合体基因CSN 6的敲除与鉴定

中国真菌学杂志　 2014, Vol. 9 　Issue (6): 321-324,330.

论著下一篇

新生隐球菌COP9复合体基因CSN 6的敲除与鉴定

杨雅骊, 张超, 孟云芳, 法振宗, 周兆婧, 赵静宇, 方伟, 廖万清

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第二军医大学附属长征医院皮肤科, 上海 200003

收稿日期:2014-10-11 出版日期:2014-12-28 发布日期:2014-12-28
通讯作者: 廖万清,E-mail:liaowanqing@sohu.com;方伟,E-mail:weifang081782@gmail.com E-mail:liaowanqing@sohu.com;weifang081782@gmail.com
作者简介:杨雅骊,女 (汉族),博士研究生在读,主治医师.E-mail:ky7812@126.com
基金资助:
国家973项目 (2013CB531601);上海市自然科学基金 (12ZR1454400,12JC1411000)

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

YANG Ya-li, ZHANG Chao, MENG Yun-fang, FA Zhen-zong, ZHOU Zhao-jing, ZHAO Jing-yu, FANG Wei, LIAO Wan-qing

Shanghai Key Laboratory of Molecular Medical Mycology and PLA Key Laboratory of Fungal Diseases, Department of Dermatology, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China

Received:2014-10-11 Online:2014-12-28 Published:2014-12-28

摘要/Abstract

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CSN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CSN 6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csn 6。结论 COP9复合体亚基CSN 6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。

关键词: 新生隐球菌, COP9信号复合体, CSN6, 致病机制

Abstract: 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 CSN 6 via overlap 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 sequencing.Results CSN 6 deletion strains were successfully constructed.Conclusions This study laid the groundwork for following functional studies of COP9/signalosome in Cryptococcus neoformans.

Key words: Cryptococcus neoformans, COP9/signalosome, CSN6, pathogenesis

中图分类号:

R379.5

杨雅骊, 张超, 孟云芳, 法振宗, 周兆婧, 赵静宇, 方伟, 廖万清. 新生隐球菌COP9复合体基因CSN 6的敲除与鉴定[J]. 中国真菌学杂志, 2014, 9(6): 321-324,330.

YANG Ya-li, ZHANG Chao, MENG Yun-fang, FA Zhen-zong, ZHOU Zhao-jing, ZHAO Jing-yu, FANG Wei, LIAO Wan-qing. Construction of knockout strains of The COP9 signalosome subunit 6 (CSN6) in Cryptococcus neoformans[J]. Chinese Journal of Mycology, 2014, 9(6): 321-324,330.

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新生隐球菌COP9复合体基因CSN 6的敲除与鉴定

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

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