鹅源草酸青霉CBHⅠ基因克隆及真核表达载体构建

Cloning of the CBHI Gene of Goose-Origin Penicillium oxalicum Currie & Thom and Its Eukaryotic Expression Vector Construction

【目的】鹅源草酸青霉F67(Penicillium oxalicum Currie & Thom)纤维素酶二糖水解酶(cellobiohydrolase,CBHⅠ)基因克隆并构建真核表达载体,为该菌株分子特性的进一步研究及构建高效纤维素分解菌奠定基础。【方法】首先通过兼并PCR法扩增CBHⅠ基因片段,然后利用改良热不对称交错PCR(TAIL-PCR)技术克隆CBHⅠ基因5′端和3′端侧翼序列,最后采用RT-PCR法扩增鹅源草酸青霉F67CBHⅠ基因序列全长,并对该基因进行生物信息学分析,构建pPIC9K真核表达载体。【结果】分别扩增到鹅源草酸青霉F67纤维素酶CBHⅠ基因片段A(EU574736)、5′端序列B1(EU603295)、3′端序列B2(EU652768)及全长基因C(EU727171),并成功构建真核表达载体pPIC9K-CBHⅠ;生物信息学分析表明,该基因蛋白序列与微紫青霉氨基酸序列同源性最高,达76%,前26个氨基酸为信号肽序列,疏水性可达2.63,由催化功能域、衔接区和真菌性纤维素结合域构成,其三级结构主要为β-sheet。【结论】鹅源草酸青霉纤维素酶CBHⅠ基因全长序列的克隆进一步丰富了丝状真菌的生物信息学资源;其真核表达载体的构建为将该菌株进一步在真核宿主中表达,从而获得高效工程菌株奠定了基础。

[Objective] The aim of the study was to clone the cellobiohydrolase gene （CBH Ⅰ ） of goose-origin PeniciUium oxalicum Currie ＆ Thorn F67 and construct the eukaryotic expression vector for the gene. [Method] A fragment of the CBH Ⅰ gene was amplified by degenerate PCR, the 5＇and 3＇ flanking sequences of CBH Ⅰ gene were cloned using TAIL-PCR, and the full-length sequence of the CBH Ⅰ 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 CBH Ⅰ gene were cloned successfully and an eukaryotic expression vector named pPIC9K-CBH Ⅰ was constructed for the gene. The bioinformatics analysis showed Penicillium janthinellum, which reached up to 76%. The first 26 that the gene had the highest homology with the gene of amino acids might be a signal peptide sequence with the hydrophobicity of up to 2.63. The CBH Ⅰ 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 CBH Ⅰ gene of goose-origin Penicillium oxalicum Currie ＆ Thorn F67 enriched the biological information resource of filamentous fungi, and the construction of the eukaryotic expression vector pPIC9K-CBH Ⅰ has laid a foundation for the expression of the CBH Ⅰ gene in eukaryotic hosts and the availability of high-performance engineering strains.