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

Abstract:

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.