| Objective Aspergillus fumigatus is an important opportunistic fungal pathogen, its growth and metabolism play an important role in pathogenicity. In fungus, CreB is involved in the system that regulates carbon catabolite repression and resistance to molybdate in Aspergillus nidulans.The function of the A.fumigatus homologue is unknown. In this study, we constructed a mutant of A.fumigatus that lacks the creB gene via split-marker recombination strategy, and its function was analyzed.Methods The method of ClusterW alignment was applied to multiple alignments of amino acid sequences. We constructed disruptant fragments for creB via split-marker recombination strategy and used PEG-mediated protoplast transformation. PCR and seqencing were used to screen for and identify homologous recombination. The phenotype studies were carried out by morphology observation for colony and analysis of resistance to molybdate.Results Alignment of the proteins indicated that CreB had the typical six UBP domains. We constructed two fragments to delete creB gene. After transformation,25hygromycin-resistant colonies were obtained. Further screened via PCR analysis and sequencing, two transformants were identified to be creB gene disruptant. Our primary phenotype studies demonstrate that the deletion of creB resulted in increased resistance to molybdate compared with the wild-type strain, which is consistent with the results in A. nidulans creB mutants.Conclusion In this study, a creB gene deletion mutant of A.fumigatus was successfully constructed via Split-Marker recombination strategy. Phenotype studies demonstrate that the deletion of creB resulted in increased resistance to molybdate compared with the wild-type strain. Structural analysis revealed CreB had the typical six UBP domains. The study provided a foundation for further studies on the molecular basis of A.fumigatus metabolism and pathogenesis. |
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