粘毛黄芩CHS基因的克隆分析及其正反义植物表达载体的构建

Cloning of Scutellaria viscidula CHS Gene and Construction of Its Sense and Antisense Plant Expression Vectors

目的:获得粘毛黄芩查尔酮合成酶(CHS)基因正反义植物表达载体,通过转化粘毛黄芩进一步探讨CHS在黄酮化合物生物合成途径上的作用机理。方法:通过RT-PCR从粘毛黄芩总RNA扩增出CHS基因目的片段,克隆至PMD18-T载体,测序并进行生物信息学分析。结果:测序证明序列正确,同时推测出CHS氨基酸序列及三维结构。克隆片段和pCAMBIA1304+载体用BlgII和BstE II双酶切,然后连接CHS基因目的片段及pCAM-BIA1304+载体大骨架片段,得到CHS正反义植物表达载体。结论:成功构建粘毛黄芩CHS基因正反义植物表达载体,并通过冻融法转化C58C1和LBA4404,为农杆菌介导的粘毛黄芩CHS基因对植物的遗传转化奠定基础。

Objective：To get Scutellaria viscidula CHS gene sense and antisense plant expression vectors,and explore the mechanism of CHS ′s effect on the flavonoid biosynthetic pathway through the transformation of Scutellaria viscidula.Methods： CHS gene fragment was amplified by Reverse Transcription Polymerase Chain Reaction（RT-PCR）from total RNA of Scutellaria viscidula and cloned into PMD18-T vector,sequenced and bioinformaticly analyzed.Results： The DNA sequence was correct and we simultaneously guessed the amino acid sequence and the three-dimensional structure of CHS.Cloned fragments and pCAMBIA1304＋ vector were digested using Blg II and BstE II at the same time,then they were connected with CHS gene fragment and pCAMBIA1304＋ big skeleton fragment to get CHS sense and antisense plant expression vectors.Conclusion： The successful construction of Scutellaria viscidula CHS gene sense and antisense plant expression vectors,and the transformation into C58C1 and LBA4404 through the freeze-thaw method,lay the foundation for Agrobacterium-mediated transformation of Scutellaria viscidula CHS gene in plant heredity.