利用iCODEHOP设计简并引物克隆益生菌FGM通透酶基因片段

The Cloning of Permease Gene Fragment from Probiotics FGM Using Degenerate Primers Designed with iCODEHOP

作者通过iCODEHOP在线设计细菌通透酶的简并引物,以发酵黄芪菌FGM基因组DNA为模板进行TouchdownPCR扩增,得到740 bp PCR产物,将产物经pGEM-T Easy载体连接,转化至JM109中,筛选阳性株并测序。序列通过BLAST x检索与GenBank进行同源性比对后,结果表明,此DNA产物序列与其他菌属来源的通透酶蛋白序列具有相似性,所克隆的序列即为FGM通透酶基因片段。用iCODEHOP在线设计的简并引物可信性强,阳性率高。FGM通透酶基因的成功克隆为细菌发酵黄芪机理研究提供了依据。

Degenerate PCR for permease using iCODEHOP to design the degenerate primers,chosing a pair of degenerate primers and utilizing the probiotics bacteria genome DNA as template.740 bp PCR product was obtained,transformed into the E.coli JM109 through being linked with pGEM-T easy vector and sequenced after filtration.Similarity alignment showed that the products of the cloned DNA were similar to those of permease gene from the other strains.The cloned sequence was putatively permease gene DNA fragment from FGM strain.The results indicated that the degenerate primers designed by the iCODEHOP software could be used to obtain specific gene fragment.Cloning of gene fragment would give a scientific warrant for further study of the fermenting mechanism of astragalus.