Split-marker 重组技术构建泛素C 末端水解酶基因缺失菌株

目的:对烟曲霉(Aspergillus fumigatus)泛素末端水解酶(creB)基因进行敲除。方法:通过氨基酸序列分析软件初步分析烟曲霉CreB蛋白结构.利用split-marker重组技术构建重组片段,并通过PEG-原生质体方法对烟曲霉野生菌株进行转化,采用PCR方法对转化子进行筛选,最后选取初步筛选的转化子进行测序鉴定。结果:结构分析显示烟曲霉CreB蛋白具有泛素特异蛋白酶(ubiquitin-processing protease)UBP亚家族六个结构域。本实验构建了转化片段并转化,在抗性平板中获得了25个Hyg抗性转化子,进一步采用PCR方法筛选到20个转化子,最终通过测序分析获得一株creB基因缺失菌株。结论:Split-marker重组技术是对烟曲霉creB基因进行敲除的快速有效的方法。获得的creB缺失菌株可用于基因功能研究。

Construction of Ubiqutin C-terminal Hydrolase (creB) Gene Deletion Mutant Via split-Marker Strategy

Objective: To construct a mutant of A.fumigatus that lacks the ubiqutin C-terminal hydrolase(creB) gene gene.Methods: The methods 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 seqenceing were used to screening and identification of homologous recombination.Results: Alignment of the proteins indicated that CreB had the typical six UBP domains.We constructed two fragments to delete creB gene and transformation,yielded 25 hygromycin-resistant colonies.20 hygromycin-resistant transformants were identified by PCR analysis.Further analysis,one strain transformant was identified to be creB gene disruptant by sequencing.Conclusion: Split-marker recombination for rapid and efficient targeted deletion of A.fumigatus creB gene.The successfully constructed creB gene deletion mutant of A.fumigatus was provided the possibilities to further analysis of the gene function.