【摘要】 目的:Aspergillus niger(CU-1)菌株α-葡萄糖苷酶基因(agdA)克隆并对其序列进行分析,构建该基因的真核表达载体。方法:设计合成的一对特异性引物,采用PCR以总DNA为模板,扩增得到DNA片段(D1);采用RT-PCR方法扩增得到DNA片段(D2)。将DNA片段D1和D2转入大肠杆菌中并进行了序列测定,序列进行BLAST比对分析。将α-葡萄糖苷酶基因的cDNA片段与表达载体pGAPZαA连接,构建重组表达载体。结果:基因agdA大小为3 127bp,含有3个外显子和4个内含子。该基因的cDNA序列大小为2 958bp,包含完整的编码框,编码985个氨基酸。agdA基因序列与已发表的α-葡萄糖苷基因序列同源性达99%,其中有6个位置的碱基发生了变化。已成功构建重组表达载体pGAPZαA-agdA。结论:Aspergillus niger(CU-1)菌株α-葡萄糖苷酶基因序列分析及重组表达载体pGAPZαA-agdA的构建为在毕赤酵母中表达Aspergillus niger(CU-1)菌株的α-葡萄糖苷酶奠定基础。更多还原

【Abstract】 Objective:Sequence analysis of the gene encoding α-glucosidase(agdA) from Aspergillus niger(CU-1) and construction of its Eukaryotic expression vector.Method:A pair of specific primers were designed and synthesized.Using the genome DNA as template,the DNA fragment(D1)was amplified with PCR,and using the total RNA as template,the DNA fragment(D2)was amplified with RT-PCR.The two DNA fragment was cloned into Escherichia coli DH5α and sequenced.Sequence analysis of the gene was on BLAST.The cDNA of agdA was inserted to eukaryotic expression vector pGAPZαA to construct the recombinant α-glucosidase expression vector.Result:The agdA was 3 127bp,composed of three exons and four introns.The cDNA of agdA was 2 958bp,containing the complete coding frame,encoding 985 amino acids.The sequence of agdA was compared with the sequence of template gene,of which the location of 6 bases had changed.The result showed that the homology could reach 99%.The eukaryotic expression vector pGAPZαA-agdA had been constructed.Conclusion: Sequence analysis of the gene agdA from Aspergillus niger(CU-1) and construction of its Eukaryotic expression vector had layed a foundation for expressing α-glucosidase of Aspergillus niger(CU-1) in Pichia pastoris.更多还原