NtSKP1-GFP植物表达载体的构建及亚细胞定位

Construction of NtSKP1-GFP Plant Expression Vector and Subcellular Location

用PCR技术扩增NtSKP1基因的编码区,定向克隆至表达载体pCAMBIA1302上构建用于瞬时表达NtSKP1蛋白的重组质粒。重组质粒经PCR和测序鉴定后用冻融法转入农杆菌LBA4404,进而经农杆菌LBA4404介导转入烟草悬浮细胞,激光共聚焦显微镜观察确定其亚细胞定位。测序结果表明,插入片段与预期序列完全一致,与载体形成了一个完整的基因表达盒;亚细胞定位结果表明NtSKP1蛋白在胞浆和核部位均有分布。

To construction plant expression vector of NtSKP1, the open reading frame of NtSKP1 was amplified by PCR, and was inserted to plasmid pCAMBIA1302. DNA sequencing showed that the sequence of the recombinant plasmid was correct. Then the recombinant plasmid was transferred into Agrobacteriurn turnefaciens strain LBA4404 through freeze-thaw method. To subcellular location the NtSKP1, the recombinant plasmid was transformed into the tobacco suspension cells mediated by Agrobacterium tumefaciens strain LBA4404, and the location of NtSKP1 was detected in both cytoplasm and nucleus.