β-1,3-1,4-葡聚糖酶基因的克隆与序列分析

Cloning and Sequence Analysis of β-1,3-1,4-Glucanase Gene

依据GenBank中收录的解淀粉芽孢杆菌(Bacillus amyloliquefaciens)β-1,3-1,4葡-聚糖酶基因序列,设计特异性引物,以提取的解淀粉芽孢杆菌基因组DNA为模板,利用聚合酶链式反应(PCR)扩增获得758 bp的β-1,3-1,4-葡聚糖酶基因(bgl),将此目的基因克隆至pTG19-T Easy载体中,经PCR、限制性内切酶鉴定和克隆片段的序列测定、比较,结果表明该克隆片段扩增准确、可靠。序列比较发现,此片段与解淀粉芽孢杆菌(B.amyloliquefacines,M15674)、枯草芽孢杆菌(B.subtilis,D00518)和地衣芽孢杆菌(B.licheniformis,AY365256)分别有99%、95%和94%的同源性。

Based on the sequence alignment of Bacillus amyloliquefaciens,two pairs of primers were designed and synthesized in this study.Using the total DNA of Bacillus amyloliquefaciens as template,the gene of β-1,3-1,4-glucanase（bgl） was amplified by PCR,which has about 750 bp in length.The gene was cloned into pTG19-T Easy vector,and the recombinant plasmid was identified by PCR,restriction enzyme analysis and sequencing.The homology analysis revealed that the nucleotide sequences of the cloned β-1,3-1,4-glucanase gene similar to the B.amyloliquefacines（M15674）,B.subtilis（D00518） and B.licheniformis（AY365256）were 99%,95% and 94%,respectively.