【摘要】 利用基因工程技术 ,将分别克隆在两个不同载体上的甜味蛋白 thaum atin c DNA基因片段连接成一个完整的 c DNA基因 ,并将该基因克隆进 p BI12 1,构建成表达载体 p BI12 1- tha.通过冻融法导入农杆菌 ,农杆菌介导叶盘法转入烟草 ,经过组培 ,得到转基因的植株 .提取转基因烟草总 DNA,经 PCR,PCR- Southern和 Southern杂交证实 ,甜味蛋白基因已整合到烟草基因组中 .RT- PCR结果证明 ,thaumatin基因已在转基因烟草中转录成 m RNA,但SDS- PAGE和甜味尝试都表明 thaumatin基因在转基因烟草中没有表达出甜味蛋白更多还原

【Abstract】 Two fragments of thaumatin cDNA were cloned in different vectors.By use of genetic technology,the two fragments were ligated into a complete thaumatin cDNA and cloned into the vector pBI121,resulting in an expression vector pBI121. After successful construction of pBI121-tha ,the recombinant plasmid was transformed into the tobacco via Agrobacterium LBA4404.The total DNA of transformed tobacco was extracted.It was confirmed that thaumatin cDNA gene has integrated into the genome of transgenic tobacco by PCR,PCR-Southern and Southern blot analysis.The RT-PCR result confirmed that the thaumatin cDNA gene was transcripted into mRNA in transformed tobacco.The SDS-PAGE analysis and sweet-testing,however,showed no difference in transformed and untransformed tobacco.These results revealed that the thaumatin cDNA gene was not translated into recombinant protein.更多还原