钝顶螺旋藻luxAB载体的构建

Construction of pUCΩluxAB vector of Spirulina platensis

实验拟构建钝顶螺旋藻luxAB载体,为螺旋藻遗传转化操作系统的建立提供技术参考和支持。使用EcoRI和SmaI双酶切质粒pUCΩGUS,胶回收获得含有Ubil启动子基因及amp基因的载体大片段;根据质粒pRL1063a中luxAB基因的序列设计引物,以质粒pRL1063a为模板(SalI酶切),PCR扩增luxAB基因片段;在T4 DNA连接酶的作用下将载体大片段和luxAB基因片段进行体外连接重组并转化感受态细胞,构建成新型质粒载体pUCΩluxAB。

In this experiment,luxAB vector of Spirulina platensis for the genetic handle system of Spirulina platensis was constructed.The plasmid pUCΩGUS was digested by the restriction enzyme SmaI and EcoRI,and the fragment with promoter Ubil and amp gene was recovered.According to the sequence of luxAB gene in the plasmid pRL1063 a,primers were designed with the digested points of SmaI and EcoRI in the upriver and downriver.With the template of pRL1063a digested by SalI,the luxAB gene fragment was received by PCR.Then the victor fragments and luxAB gene were recombined with T4 DNA ligase.The reconstructed plasmid is pUCΩluxAB with the luxAB and amp gene.Furthermore,the reconstructed plasmid was transformed into E.coliDH5α.