抗TMV、CMV转基因烟草的初步选育

The studies on transgenic tobacco resistant to TMV and CMV

将烟草花叶病毒复制酶54 KD蛋白基因构建到质粒pSSZ32.54K,导入根癌农杆菌EHA105株系,用农杆菌介导的叶盘法转化含CMV CP基因的纯合系烟草,获得了含CMV CP基因和TMV RepE基因的转基因烟草,以及只含TMV RepE基因的转基因烟草。以繁殖于新鲜普通烟叶片上的TMV为毒源,机械摩擦接种含双基因的烟草和只含CMV CP基因的烟草。接种15 d后,观察植株发病情况,发现转双基因的烟草无花叶症状出现,而对照植株(只含CMV CP基因)出现花叶症状。通过酶联免疫吸附试验(ELISA)检测病毒含量,发现转基因植株体内无病毒积累,而对照植株体内有病毒积累。因此,含RepE基因的烟草对TMV有一定抗性。

For the purpose to obtain TMV and CMV-resistant transgenic tobacco plants,the tobacco mosaic virus replicase（RepE） 54KD protein gene was cloned into plant expression vector pSSZ32.54K and the recombined pSSZ32.54K was mobilized into Agrobacterium tumefaciens strain EHA105.Leaf discs of homozygous tobacco containing CMV-CP gene were transformed with A.tumefaciens.Transgenic tobacco plants containing RepE gene and CP gene were obtained.Trangenic plants only containing RepE gene were obtained.Transgenic plants with RepE and CP genes and 3 untransgenic control plants were inoculated with TMV by mechanical inoculation.The control plants emerged mosaic symptom,but the transgenic plants did not show any mosaic symptom.The TMV accumulation of transgenic plants was remarkably lower than untransgenic plants by ELISA.The result indicated that the transgenic tobacco plants mediated by RepE gene were resistant to TMV infection.