Construction of a new vector for targeted gene disruption in Candida albicans
10.3760/cma.j.issn.1000-6680.2009.02.001
- VernacularTitle:一种用于白念珠菌基因敲除的新型载体的构建
- Author:
Hui HU
;
Yongxin ZHANG
- Publication Type:Journal Article
- Keywords:
Candida albicans;
Zeoein;
Gene,knockout techniques;
Microbial sensitivity tests;
Recombination,genetis
- From:
Chinese Journal of Infectious Diseases
2009;27(2):65-68
- CountryChina
- Language:Chinese
-
Abstract:
Objective To explore the feasibility of using Zeocin resistance as a new positive selection marker for the genetic manipulation of Candida albicans (C.albicans).Methods The susceptibility of C.albicans strain CAI4 to Zeocin was determined using a broth microdilution method in pH range from 6.0 to 8.0.The ZeoR expression cassette was amplified from plasmid pGAPZ-α-A by polymerase chain reaction (PCR).An integrated vector used to knockout AAF1 gene was constructed by gene splicing.Then the C.albicans CAI4 was transformed with this integrated plasmid containing the ZeoR expression cassette using lithium chloride method and the target AAF1 gene was replaced.The recombinant C.albicans was screened and the genome was extracted and amplified by PCR with two pairs of primers,then confirmed using genetic method.Results The C.albicans CAI4 was sensitive to Zeocin in pH range from 6.0 to 8.0.And the concentration of Zeoein(100 mg/L) at pH 7.0 was used to select recombined C.albicans.The recombined integrated vector was confirmed by DNA sequencing.The ZeoR expression cassette was detected using PCR method in recombined C.albicans.The AAF1 gene was replaced by ZeoR gene.Conclusion A new vector for targeted gene disruption in C.albicans is successfully constructed.
