马尾松GGPPS基因cDNA序列克隆及正义、反义植物表达载体的构建

Cloning of Pinus massoniana GGPPS cDNA Fragment and Constructing of Sense and Antisense Expression Vectors

以马尾松幼苗针叶组织为材料,依据Genbank中已报道的GGPPS基因CDS区序列设计特异引物,经RT-PCR技术克隆得到GGPPS基因cDNA序列。克隆片段经酶解后分别正向、反向插入植物表达载体p-GFP,构建成GGPPS基因正义、反义植物表达载体。采用冻融法将重组质粒导入农杆菌菌株LBA4404,建立植物表达载体系统,为下一步植物转化研究过表达、抑制表达GGPPS基因对萜烯类物质合成的影响建立研究基础。

The cDNA nucleotide sequence of GGPPS was cloned by Reverse Transcription Polymerase Chain Reaction （RT-PCR） from needles tissue of Pinus massoniana seedling. The clone primers were desgined with two enzyme recognition sites. The cloned cDNA of GGPPS gene was digested and further introduced into binary vector p-GFP in forward and reverse orientation respectively, which gived an express plasmids containing the sense GGPPS gene or the antisense GGPPS gene respectively. Then the recombinant plasmids were transferred into Agrobacterium tumefaciens LBA4404 by freezing and thawing method, so the plant expression vector systems were established. This study provided the basis for the further plant transformation research about the impact of GGPPS gene enhance expression or suppression expression on terpene chemicals synthesis.