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鹅源草酸青霉CBHⅠ基因克隆及真核表达载体构建
引用本文:王宝维,张倩,葛文华,张名爱,岳斌,黄国清.鹅源草酸青霉CBHⅠ基因克隆及真核表达载体构建[J].中国农业科学,2010,43(7):1464-1472.
作者姓名:王宝维  张倩  葛文华  张名爱  岳斌  黄国清
作者单位:(青岛农业大学优质水禽研究所)
基金项目:国家科技支撑计划,山东省农业良种产业化工程项目 
摘    要:【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。

关 键 词:鹅源草酸青霉  外切葡聚糖酶  TAIL-PCR
收稿时间:2009-08-17;

Cloning of the CBHI Gene of Goose-Origin Penicillium oxalicum Currie & Thom and Its Eukaryotic Expression Vector Construction
WANG Bao-wei,ZHANG Qian,GE Wen-hua,ZHANG Ming-ai,YUE Bin,HUANG Guo-qing.Cloning of the CBHI Gene of Goose-Origin Penicillium oxalicum Currie & Thom and Its Eukaryotic Expression Vector Construction[J].Scientia Agricultura Sinica,2010,43(7):1464-1472.
Authors:WANG Bao-wei  ZHANG Qian  GE Wen-hua  ZHANG Ming-ai  YUE Bin  HUANG Guo-qing
Affiliation:WANG Bao-wei1,2,ZHANG Qian1,GE Wen-hua1,ZHANG Ming-ai2,YUE Bin1,HUANG Guo-qing2 (1High Quality Waterfowl Research Institute,Qingdao Agricultural University,Qingdao 266109,Sh,ong,2College of Food Science , Engineering,Sh,ong)
Abstract:Objective] The aim of the study was to clone the cellobiohydrolase gene (CBH Ⅰ) of goose-origin Penicillium 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 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 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 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.
Keywords:TAIL-PCR  goose-origin Penicillium oxalicum Currie & Thorn  TAIL-PCR
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