马尾松银松素合酶基因转化双子叶植物表达载体的构建及鉴定

Construction and Characterization of Dicotyledon Expression Vector Containing Pinosylvin Synthase Gene from Pinus massoniana

以马尾松为材料,在获得马尾松银松素合酶基因的基础上,构建了该基因转化双子叶植物的表达载体,该表达载体含有35S启动子和NOS终止子,能启动基因在双子叶植物中高效地表达.设计并合成分别含有Bgl和BstE酶切位点的上下游引物,通过PCR反应扩增出含有该酶切位点的银松素合酶基因cDNA全序列,酶切后连接到pCAMBIA1301植物表达载体上.经PCR鉴定、酶切分析及DNA测序证实cDNA片段大小、序列以及读码框的正确性.最后通过电击将表达载体转化农杆菌LBA4404,为进一步研究银松素合酶基因的功能奠定基础.

A dicotyledon expression vector was constructed for transforming pinosylvinsynthase gene from Pinus massoniana. This vector contains 35S promoter and NOS terminator, which can drive the gene＇s high expression in dicotyledon. Primers with Bgl Ⅱ and BstE Ⅱ were designed and PCR was used to amplify the pinosylvin synthase gene. A new cloning vector was then constructed by inserting the amplified pinosylvin synthase gene into pCAM-BIA1301 expression vector. PCR and restriction analysis showed that the inserted DNA fragment was the size of expecting. Furthermore, sequencing showed that the inserted DNA fragment was not out of frame. The expression vector was then transferred into Agrobacterium tumefaclens LBA4404, which was the foundation to function research of pinosylvin synthase.