关键词: 基因敲除, RAS1基因, SN152菌株, HIS-LEU-ARG, URA -Blaster法

Abstract: Objective: To construct RAS1 gene knockout strain in Candida albicans using HIS-LEU-ARG knocking-out strategy and URA-Blaster methodology respectively and compare the success rate of the two strategies. Methods: In the HIS-LEU-ARG knocking-out strategy, the genomic DNA of SN152 strains were amplified and fused with DNA of auxotrophic markers to construct homologous fusion fragments using fusion PCR. For URA-Blaster methodology, the genomic DNA of CAI4 strains were inserted into sides of hisG-URA3-hisG knockout cassette to construct knockout plasmid using seamless cloning. Fusion gene fragments and plasmid linearization product were then introduced into Candida albicans SN152 and CAI4, respectively. Positive colonies were screened on the nutritional defect medium and performed homologous recombination two times to knock out both alleles in the RAS1 gene. Results: The Candida albicans RAS1 double allelic deletion strains were successfully constructed using HIS-LEU-ARG knocking-out strategy. However, URA-Blaster methodology was repeated more than three times and failed to construct RAS1 double allelic deletion strains. Conclusion: HIS-LEU-ARG knocking-out strategy is more suitable for the construction of RAS1 gene knockout strain in Candida albicans than the URA-Blaster methodology.

Key words: gene knockout, RAS1 gene, SN152 strain, HIS-LEU-ARG, URA -Blaster