极地研究

极地研究 ›› 2013, Vol. 25 ›› Issue (3): 249-256.DOI: 10.3724/SP.J.2013.00249

• 研究论文 • 上一篇    下一篇

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

周莉莉1,2  陈瑞琴1,2  陈秀兰3  曾胤新2  陈波1,2   

  1.  
    1华东理工大学生物工程学院，上海 200237；
    2国家海洋局极地科学重点实验室，中国极地研究中心，上海 200136；
    3山东大学微生物技术国家重点实验室，山东 济南 250100
  • 收稿日期:2012-12-19 修回日期:2013-01-15 出版日期:2013-09-30 发布日期:2013-09-30
  • 通讯作者: 周莉莉

CLONING OF THE β-GALACTOSIDASE GENES FROM MARINOMONAS SP. BSI20584 by hiTAIL-PCR

Zhou Lili1,2，Chen Ruiqin1,2，Chen Xiulan3，Zeng Yinxin2，Chen Bo1,2   

  1. 1School of Biotechnology of East China University of Science and Technology, Shanghai 200237，China; 2Polar Research Institute of China，Key Laboratory for Polar Science, SOA, Shanghai 200136，China;3State Key Laboratory of Microbial Technology, Jinan 250100
  • Received:2012-12-19 Revised:2013-01-15 Online:2013-09-30 Published:2013-09-30

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摘要： 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。

关键词: β-半乳糖苷酶, 高效热不对称交错PCR, 染色体步移

Abstract: A complete gene of a β-galactosidase was isolated from Marinomonas sp. BSi20584 by hiTAIL-PCR(high-efficiency thermal asymmetric interlaced). A pair of primes were designed according to online conserved regions in other β-galactosidases.The conversed fragment of β-galactosidase was gained by PCR. The N-terminal DNA sequence was amplified by PCR using primers based on N-terminal amino acid sequence of the purified enzyme. Upstream and downstream of sequences already obtained and named nbs were gained  by hiTAIL-PCR. In the end , the whole gene was amplified by primers designed according to the assembly DNA sequences. In this article the β-galactosidase gene was successfully isolated and was 1971bp. NCBI blast indicated it was a new member of glycosylhydrolase family 42.

Key words: β-galactosidase, hiTAIL-PCR, gene walking