TAIL-PCR法快速分离红曲霉色素突变株T-DNA插入位点侧翼序列

Isolation of DNA Sequence Flanking T-DNA by Thermal Asymmetric Interlaced PCR from Monascus Pigment-producing Mutants

采用热不对称交错PCR(thermal asymmetric interlaced PCR,TAIL-PCR)法分离红曲霉色素突变株T-DNA插入位点的侧翼序列,扩增到了长度介于500bp～1300bp之间的7个DNA片段,对这些DNA片段测序,并采用生物信息学方法对测序结果进行了分析,表明其中有1个片段与烟曲霉Af293发育调控子flbA的相似性较高。TAIL-PCR法成功应用于分离红曲霉突变株T-DNA插入位点的侧翼序列,为大规模获取插入位点侧翼序列建立了可行的方法,也为进一步研究红曲霉功能基因奠定了基础。

In this paper, thermal asymmetric interlaced PCR （TAIL-PCR） was adopted to amplify DNA sequences flanking T-DNA of pigment-producing mutants of Monascus spp. Seven DNA fragment which renged from 500bp to 1300bp were isolated. Then the fragment were sequenced and their functions were analyzed with Blast tool on line supplied by NCBI. The results showed that one of the DNA sequences was of similarity to the nucleotide sequence was of similarity to the necleotide sequence ceding for the developmental regulator offlbA from Aspergillusfumigatus Af293 and the similarity of them amounted to 82%. The successful amplification to Monascus spp. by TAIL-PCR provides an efficient method to isolate DNA sequences flanking T-DNA from the monascus insertional mutants on a large scale and will be beneficial to investigate the functional gene of Monascus spp.