一种强力霉素诱导型白念珠菌基因敲除与筛选标记再循环工具包

摘要

[Objective] To construct a fast,convenient and economical DNA manipulating toolkit for gene deletion and marker recycling in Candida albicans.[Methods] By using Exo III-mediated ligase-independent cloning strategy,loxP sites were inserted into the flanking regions of heterologous selection markers CmLEU2,CdHIS1,and CdARG4,yielding the templates for marker cassettes amplification.The Tet-on promoter was assembled using a rTetR element which was codon-optimized and chemically synthesized.Codon-optimized recombinase Cre was placed downstream of the Tet-on promoter.Then this cassette was inserted into the CDS region of selection markers CdHIS1 and CdARG4,generating vectors for marker recycling.[Results] We constructed three loxP-flanked marker gene-containing vectors for gene deletion in C.albicans,and two vectors for marker recycling,containing the recombinase gene Cre under the control of the Tet-on promoter.[Conclusion] We successfully constructed an inducible gene deletion and marker recycling vector system in C.albicans,which was practically applied in gene deletion strain constructions.This system is helpful to construct single and multiple gene deletion strain constructions.%[目的]在白念珠菌中建立一个快捷方便经济的基因敲除与筛选标记再循环的DNA操作系统.[方法]通过Exo III介导的不依赖于连接酶的克隆策略,在异源筛选标记基因CmLEU2、CdHIS1和CdARG4基因的两侧分别插入了loxP位点,成为筛选标记基因盒扩增的模板.全基因合成了经过白念珠菌密码子优化的rTetR元件,并组装成Tet-on启动子.将密码子优化的重组酶Cre基因置于该启动子控制下.然后将他们插入筛选标记基因CdHIS1和CdARG4的CDS区域,形成筛选标记基因再循环载体.[结果]构建了3个用于白念珠菌基因敲除的侧翼含有loxP位点的筛选标记基因载体,以及2个含有Tet-on启动子控制的Cre酶的载体用于筛选标记基因的再循环.[结论]成功构建了一个白念珠菌中可诱导的基因敲除和筛选标记再循环的载体系统并成功应用于多个基因缺失株构建.这个系统有助于快速构建白念珠菌的单基因和多基因敲除菌株.