关键词: 酿酒酵母, YVC1基因, Cre/loxP系统, 基因敲除, 等位基因

Abstract: The calcium ion channel of Yvc1 on the vacuolar membrane inSaccharomyces cerevisiaeis one of the important ways to regulate the intracellular Ca2+ concentration. As a second messenger, Ca2+ can improve the ethanol production capacity ofSaccharomyces cerevisiae. Therefore, it will lay the foundation for the further study of calcium channel function that theYVC1gene deletion strain is constructed. The presence of theYVC1gene inSaccharomyces cerevisiaeDL5168 was confirmed by PCR. The chromosome ploidy of strain DL5168 was identified by Ziehl-Neelsen’s stain and genome-specific PCR. Based on the principle of Cre/loxP system,YVC1gene knockout module was constructed and transformed into the strain of DL5168 via lithium acetate conversion method, and positive recombiners were screened on YPD medium supplemented with 100 μg/mL G418. The pSH65 plasmid was transferred into the positive recombinant to remove the resistance gene ofKanMX. The results showed that the genome ofSaccharomyces cerevisiaeDL5168 contained the vacuole conductivity-1 geneYVC1. Strain DL5168 was proved to be an aα-type diploid. The pUG6-YVC1-loxP-KanMX knockout module successfully underwent homologous recombination with DL5168, yielding positive recombinants. The resistance geneKanMXon the recombinant DL5168-yvc1Δ-kanmxwas successfully removed with the pSH65 plasmid producing Cre recombinase under the induction of galactose. This resulted in the generation of aYVC1deficient allele strain, designated DL5168-yvc1Δ. The allele ofYVC1on the other homologous chromosome was deleted by the same method. Finally, theYVC1gene diploidy deletion strain of DL5168-yvc1Δ/Δ was successfully constructed in this study.

Key words: Saccharomyces cerevisiae, YVC1gene, Cre/loxP system, gene knockout, allele