玉米C_4型PPDK基因的分段PCR克隆及其表达载体构建

Segmented-PCR Cloning of C_4 PPDK from Maize and Construction of Its Expression Vector

丙酮酸磷酸双激酶(PPDK)是C4植物和景天科酸代谢(CAM)植物光合作用的关键酶,催化形成固定CO2的初始分子受体磷酸烯醇式丙酮酸(PEP)。玉米的C4型PPDK基因序列较长,难以直接克隆。本研究设计多对引物,首先利用常规PCR扩增PPDK启动子,再利用分段PCR扩增PPDK基因全长编码序列;测序证实克隆序列正确无误,最后利用In-Fusion技术将各个片段定向克隆至载体p CAMBIA-1391Z,成功构建了玉米C4型PPDK基因的植物表达载体。最终为C4型PPDK基因功能研究和C3植物遗传转化提供可用的基因序列,为全长基因的高效克隆及表达载体构建提供参考。

Pyruvate, orthophosphate dikinase(PPDK) is a cardinal enzyme of the C4 and CAM pathway. Its role in C4 photosynthesis is to catalyze the regeneration of the CO2 acceptor PEP. The C4 PPDK gene's sequence in maize is long and is very difficult to clone by conventional method. In this study, some primers were designed to cloning the C4 PEPC gene's promoter and full-length sequence by conventional PCR and Segmented-PCR,and transferred fragments into the vector p CAMBIA-1391 z using In-Fusion technology after sequencing. Finally the p CAMBIA-1391Z-ppdk was gotten plant expression vector. The successful cloning of the gene has set the base for C4 PPDK functions research and provide available genetic sequences for C3 plants genetic transformation. It also provides an easible method to clone long-strand DNA and Construction of its expression vector.