关键词: 鹅源草酸青霉, 外切葡聚糖酶, TAIL-PCR

Abstract:

【Objective】 The aim of the study was to clone the cellobiohydrolase gene (CBHⅠ) of goose-origin Penicillium oxalicum Currie ＆ Thom F67 and construct the eukaryotic expression vector for the gene. 【Method】 A fragment of the CBHⅠ gene was amplified by degenerate PCR, the 5′and 3′ flanking sequences of CBHⅠ gene were cloned using TAIL-PCR, and the full-length sequence of the CBHⅠ gene was cloned by RT-PCR. The construction of the eukaryotic expression vector for the gene was also studied. 【Result】 Fragment A (EU574736), 5′ sequence B1 (EU603295), 3′ sequence B2 (EU652768) and full-length sequence C (EU727171) of the CBHⅠ gene were cloned successfully and an eukaryotic expression vector named pPIC9K-CBHⅠ was constructed for the gene. The bioinformatics analysis showed that the gene had the highest homology with the gene of Penicillium janthinellum, which reached up to 76%. The first 26 amino acids might be a signal peptide sequence with the hydrophobicity of up to 2.63. The CBHⅠ gene consisted of the catalytic domain, convergence zone and cellulose-binding domain and its tertiary structure was mainly composed of β-sheets. 【Conclusion】 The successful cloning of the full-length sequence of CBHⅠ gene of goose-origin Penicillium oxalicum Currie ＆ Thom F67 enriched the biological information resource of filamentous fungi, and the construction of the eukaryotic expression vector pPIC9K-CBHⅠ has laid a foundation for the expression of the CBHⅠ gene in eukaryotic hosts and the availability of high-performance engineering strains.

Key words: goose-origin Penicillium oxalicum Currie &, Thom, CBHⅠ, TAIL-PCR