摘要
GFP基因被广泛地应用于植物转基因以及基因功能验证研究,然而构建与GFP融合的植物表达载体,常会因酶切位点难以选择,而使得载体构建过程复杂,周期长。以含GFP的质粒pCAMBIA-1302为基础,消除此质粒本身的多克隆位点(MCS),并在此质粒GFP基因序列前插入原核表达载体pET-32b的多克隆位点,构建了植物GFP亚细胞定位载体卡盒pCEG;采用农杆菌介导的转化方法,将此载体卡盒pCEG导入模式植物烟草叶片中进行瞬时表达,用激光共聚焦显微镜观察GFP蛋白的亚细胞定位情况,发现GFP蛋白均匀的在细胞质和细胞核中表达,证明此载体卡盒可用于植物基因的亚细胞定位分析,并为构建目的基因与GFP融合的植物表达载体提供了很多可用的酶切位点,对植物基因功能验证提供了分子操作简便的植物表达载体卡盒,具有重要的利用价值。
GFP gene was widely applied in the transgenic plants and genes function＇s proving study.However,the construction of GFP fused plant expression vectors was always complicated and time consuming due to the hard choose of restriction enzyme cutting sites.Use plasmid pCAMBIA-1302 which included GFP gene as a original vector,and eliminated its own multiple clone site（MCS）,and then inserted pET-32b＇s multiple clone site in front of GFP gene sequences,named the eventual plant GFP subcellular localization vector box pCEG.By Agrobacterium-mediated transformation method,pCEG instantaneous expressed in tobacco leaves,and GFP protein subcellular localization was observed by laser confocal microscope.The result displayed that the GFP proteins expressed in both cytoplasm and nucleus,this means the pCEG box can be used conveniently for plant genes subcellular localization analysis and provide a lot of usable restriction enzyme cutting site for plant genes fusioned with GFP,and the pCEG box is convenient for plant genes function analysis and has great use value.
出处
《东北农业大学学报》
CAS
CSCD
北大核心
2011年第4期83-88,共6页
Journal of Northeast Agricultural University
基金
国家高科技发展计划(863计划)(2008AA10Z153)
国家自然科学基金资助项目(30570990)
黑龙江省科技厅重大攻关项目(GA06B10)
黑龙江省教育厅科技项目(11521024)
黑龙江省教育厅科技项目(11521021)
关键词
载体卡盒构建
亚细胞定位
GFP
烟草
construction of vector box
subcellular localization
GFP
tobacco
