不同筛选标记法构建白念珠菌RAS1基因敲除株的比较

Comparison of knocking-out the RAS1 gene in Candida albicans using different auxotrophic markers

目的采用两种筛选标记法(HIS-LEU-ARG基因敲除策略和URA-Blaster法)分别构建白念珠菌RAS1基因敲除株,并对两种方法的成功率进行比较。方法 HIS-LEU-ARG基因敲除策略中,以SN152菌株基因组DNA、筛选标记DNA为模板,采用融合PCR技术构建融合基因片段。URA-Blaster法中,以CAI4基因组DNA、hisG-URA3-hisG基因敲除盒为模板,运用无缝克隆技术构建RAS1基因敲除质粒。随后,分别将上述两种方法得到的融合基因片段和质粒线性化产物转染至白念珠菌SN152、CAI4细胞内,在营养缺陷培养基上获得阳性转化子,通过2次同源重组敲除RAS1两条等位基因。结果采用HISLEU-ARG基因敲除策略成功构建RAS1双等位基因敲除株。而采用URA-Blaster方法,重复多次均未能成功构建白念珠菌RAS1双等位基因敲除株。结论 HIS-LEU-ARG基因敲除策略比URA-Blaster法更适用于RAS1基因敲除株的构建。

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 rates of the two strategies. Methods In the HIS-LEU-ARG knockingout strategy,the genomic DNA of SN152 strains was 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 was inserted into sides of hisG-URA3-hisG knockout cassette to construct knockout plasmid using seamless cloning. Fusion gene fragments and plasmid linearization products were then introduced into Candida albicans SN152 and CAI4,respectively. Positive colonies were screened on the nutritional defect medium and performed homologous recombination twice 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 repeated more than three times and failed to construct RAS1 double allelic deletion strains. Conclusion HIS-LEU-ARG knockingout strategy is more suitable for the construction of RAS1 gene knockout strain in Candida albicans than the URA-Blaster methodology.