树干毕赤酵母木糖还原酶基因的克隆及在大肠杆菌中的表达

Cloning of Xylose Reductase Gene from Pichia stipitis and Expression in E.coli

根据NCBI中的木糖还原酶基因序列设计引物,利用高保真聚合酶克隆树干毕赤酵母木糖还原酶基因,加A后克隆到质粒pGM-T中,测序验证。然后将目的基因克隆到含有强启动子的穿梭表达载体p424GPD中,构建含有XYL1基因的重组质粒p424GPD-XYL1。将p424GPD-XYL1转化到大肠杆菌中,提取总蛋白,聚丙烯酰胺凝胶电泳分析,酶活测定确定木糖还原酶基因XYL1在大肠杆菌中得到活性表达,表明表达载体构建成功。表达载体的成功构建为后续构建重组酿酒酵母利用木糖发酵奠定基础。

The primers were designed according to the sequence of XYL1 reported on the NCBI.The gene encoding xylose reductase was amplified by using high-fidelity DNA polymerase from total DNA of Pichia stipitis,and cloned into pGM-T vector after added A.The vector was verified by sequencing.The target gene was cloned into a shuttle vector p424GPD and placed under the strong constitutive promoter GPD.The recombinant vector p424GPD-XYL1 was transformed into E.coli TOP10,extracting the total protein and analysis by SDS-PAGE.The xylose reductase gene was expressed in E.coli by means of enzyme assay.The construction of this vector provides an important basis for the further establishment of recombinant Saccharomyces cerevisiae.