宇佐美曲霉中β-甘露聚糖酶基因调控序列的克隆

Cloning of the Regulatory Sequences of β-mannanase Gene from Aspergillus usamii

DOI：10.3969/j.issn.1673-1689.2012.02.006

中文关键词: 宇佐美曲霉 β-甘露聚糖酶 基因 调控序列 T载体介导的PCR

英文关键词: Aspergillus usamii β-mannanase gene regulatory sequence T vector-mediated PCR

基金项目:国家自然科学基金项目(20776061)

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中文摘要:

建立了一种利用限制性内切酶酶切、pUCm-T载体连接和巢式PCR技术,基于部分已知DNA序列测定其两侧翼未知DNA序列的新方法,命名为T载体介导的PCR技术,并将此技术应用于宇佐美曲霉(Aspergillus usamii)E001的β-甘露聚糖酶基因(Aus man5A)5′和3′端调控序列的克隆。结果表明:该PCR技术具有操作简便、引物特异性强、结果验证简捷、操作限制少等特点;借助于T载体介导的PCR技术、分子克隆和序列测定等手段,成功地测定了Ausman5A两侧翼的调控序列。

英文摘要:

In this manuscript,a novel T vector-mediated PCR technique was developed by using restriction enzyme digestion,pUCm-T vector ligation and nested PCR technique.This novel method can be used to determine two flanking unknown DNA sequences of a partial known DNA sequence.And this method was applied to clone the 5′-and 3′-end regulatory sequences of β-mannanase gene(Aus man5A) from Aspergillus usamii E001.The results showed that this method exhibited many advantages,for example,simplicity of operation,high specificity of primers,rapidness of result verification,and few restrictions for operation.By means of T vector-mediated PCR amplification,molecular cloning and DNA sequencing,the flanking regulatory sequences of the Aus man5A was successfully determined.

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