摘要
按植物偏爱密码子设计合成了228bp带his-tag(6×his)人胰岛素原基因(hmins),随后构建了植物双元表达载体p35S::hmins(骨架质粒是pCAMBIA2300),通过冻融法导入根癌农杆菌(Agrobacterium tumefaciens)EHA105制备工程菌,用农杆菌介导法转化烟草叶盘。经卡那霉素(50mg·L-1)筛选获得98株抗性植株,PCR和southern blotting分析确认获得了6株独立转基因烟草,hmins在转基因烟草基因组中的拷贝数介于2~4;RT-PCR和western blotting分析结果表明,hmins在转基因烟草中成功表达(转录和翻译水平);ELISA结果显示,hmins在转基因烟草叶片中表达量为0.5~1.1μg·g-1·FW。该研究为植物源胰岛素研制做了必要的前期准备。
According to plant codon usage bias,the human proinsulin gene(hmins,228 bp) was designed and synthesized,which includes encode sequence of his-tag(6×his),then plant binary expression vector p35S::hmins(backbone plasmid is pCAMBIA2300)was constructed.Leave explants of tobacco(Nicotiana tabacum L.cv.BaiRihong) were transformed with A.tumefaciens EHA105 harbouring plasmid of the p35S::hmins.Ninety-eight regenerated tobacco plants which resistant to kanamycin(50 mg·L-1)were obtained,and six independent transgen...
出处
《上海交通大学学报（农业科学版）》
2010年第1期14-18,52,共6页
Journal of Shanghai Jiaotong University(Agricultural Science)
基金
国家"863"计划生物反应器重大专项资助(2007AA100503)
关键词
重组人胰岛素原
转基因烟草
糖尿病
recombinant human proinsulin
transgenic tobacco
diabetes
