TAIL-PCR方法扩增稀有放线菌小单孢菌中ΦC31的attB序列研究

Amplifying attB Sequence of ΦC31 in Rare Actinomycete Micromonospora by TAIL-PCR

[目的]为了研究链霉菌噬菌体ΦC31在小单孢菌40027菌株中的整合位点序列。[方法]以20 ng整合质粒pSET152的小单孢菌40027菌株基因组DNA为模板,利用3个特异性巢式引物(PF1、PF2和PF3)与随机引物AD2,通过热不对称交错PCR(TAIL-PCR)扩增稀有放线菌小单孢菌中ΦC31的attB序列,用1%琼脂糖凝胶电泳来检测扩增产物的大小。[结果]扩增结果表明,第三轮扩增产物250 bp左右的条带比第二轮对应带略小,第二轮扩增产物对应带比第一轮对应带略小,与3个特异性巢式引物预期扩增结果相符,第三轮扩增产物250 bp左右那一条带为阳性带,经回收后,进行克隆并测序。序列分析表明:该阳性带包含ΦC31在小单孢菌40027菌株中的attB序列。[结论]TAIL-PCR方法为attB序列的克隆提供了一种简便、高效的新方法。

[Objective] The aim was constructed to study the sequence of integration sites of Streptomyces phage ΦC31 in Micromonospora sp. 40027 strain. [Method] Taking genomic DNA of Micromonospora sp.40027 strain with 20 ng integration plasmid pSET152 as a template, 3 specific nested premiers（PF1,PF2 and PF3）and random premier AD2 were used to amplify the attB sequence of ΦC31 in rare aetinomycete Micromonospora by TAIL-PCR and their products were detected by 1% agarose gel electrophoresis, [Result] The amplifying results showed that the band about 250 bp in the tertiary PCR products was smaller than that in the secondary PCR products, and that in the secondary PCR products was smaller than that in PCR products, which accorded with prospective results from 3 specific netted premiers. The band about 250 bp in the products of tertiary PCR products was positive, which was cloned and sequenced after recovering, The sequence analysis indicated that the positive band contained the attB sequence of ΦC31 in Micromonospora sp. 40027 strain. [Conclusion] TAIL-PCR provided a simple and effcient new method for the cloning of the attB sequence.