应用hiTAIL-PCR技术克隆Marinomonas sp.BSi20584菌株β-半乳糖苷酶基因

CLONING OF β-GALACTOSIDASE GENES FROM MARINOMONAS SP. BSI20584 BY HITAIL-PCR

实验使用hiTAIL-PCR(high-efficiency thermal asymmetric interlaced PCR,高效热不对称交错PCR)方法,从海单胞菌Marinomonas sp.BSi20584中克隆β-半乳糖苷酶基因。从GenBank注册的已知β-半乳糖苷酶氨基酸序列出发,设计引物克隆编码β-半乳糖苷酶保守片段的DAN序列;根据Marinomonas sp.BSi20584天然酶N端氨基酸残基测序结果,设计上游引物,保守DNA片段5'端设计下游引物,克隆N端到保守序列之间的DNA片段;将所得的2个DNA片段拼接并命名拼接后的片段为nbs。根据hiTAIL-PCR方法设计引物,染色体步移克隆nbs上下游片段,拼接上下游片段得到全长序列,设计引物扩增全长片段并测序。本文成功克隆了Marinomonas sp.BSi20584菌株β-半乳糖苷酶的编码基因,全长1 971 bp,NCBI比对表明其编码产物为GH-42家族的一个新成员。

A complete β-galactosidase gene was isolated from Marinomonas sp. BSi20584 by high-efficiency thermal asymmetric interlaced polymerase chain reaction （hiTAIL-PCR）. A pair of primers was designed according to the i- dentified conserved regions of other β-galactosidases. The conserved β-galactosidase fragment was obtained by PCR. The N-terminal DNA sequence was amplified using primers based on the N-terminal amino acid sequence of the purified enzyme. And these two sequences were assembled resulting one sequence named nbs. Upstream and downstream sequences of nbs were obtained by hiTAIL-PCR. Finally, the entire gene was amplified using primers designed according to the whole assembled DNA sequence. In this article, a 1 971 bp β-galactosidase gene was successfully isolated. An NCBI search indicated the gene was a new member of glycosylhydrolase family 42.