摘要
根据已知的杏仁、黑樱桃醇腈酶基因序列设计了一对同源引物,扩增得到枇杷醇腈酶基因的部分序列.分别设计了三条基因特异性引物和一条基因非特异性引物,采用改进的SEFA PCR方法,进行上下游未知序列的扩增,成功地获得包括启动子区、转录终止子在内的枇杷醇腈酶基因序列,全长5.5 kbp.所得醇腈酶基因含有4个外显子和3个内含子.编码的氨基酸残基序列包括25个氨基酸残基的信号肽和527个氨基酸残基的成熟酶蛋白.采用重叠延伸PCR方法将4个外显子连接获得了连续的醇腈酶编码基因,与pET27b(+)质粒成功构建表达载体,转化Rosetta(DE3).表达的醇腈酶大部分以包涵体形式存在,最终表达的酶活力为1.2 U.mL-1.
One pair of homology primers was designed based on the known gene sequences of mandelonitrile lyase from almond and black cherry.The inner partial gene sequence of loquat mandelonitrile lyase was amplified using the homology primers.Then three gene specific primers and one non-specific primer were designed for SEFA PCR method to amplify the upstream/downstream flanking sequences.5.5 kbp genomic sequence was successfully obtained,which included the promoter region,mdl gene sequence and transcription terminator.The mdl gene contained 4 exons and 3 introns.The coded MDL had 552 amino acids with 25 amino acid-long signal peptide.The mature MDL was 527 amino acids in length.The 4 exons were ligated together into a continuous coding sequence using overlapping extension PCR method,and then was constructed into recombinant expression plasmid with pET27b（＋）.Rosetta（DE3） was used as host strain for expression of MDL.The expressed MDL mainly existed in inclusion bodies.The enzyme activity reached 1.2 U·mL-1.
出处
《福州大学学报（自然科学版）》
CAS
CSCD
北大核心
2011年第6期960-964,共5页
Journal of Fuzhou University(Natural Science Edition)
