摘要
将构建的携带烟草花叶病毒（ＴＭＶ）５４ＫＤ蛋白基因及黄瓜花叶病毒（ＣＭＶ）３’端缺失０．９Ｋｂ的１．６Ｋｂ片断复制酶基因植物表达载体ｐＢＩＣＴ，导入根癌农杆菌３１１ＳＥ系，用叶圆盘法转化烟草，在含有卡那霉素的选择性培养基上诱导生芽、生根，得到２８株成活苗．移入大田后，随机挑选４．株植物，提取植物总ＤＮＡ，经ＰＣＲ扩增、Ｓｏｒｔｈｅｒｎ杂交检测，结果４株植物中都有复制酶基因的整合，而未经转化的对照组植物则没有．转基因植株在大田试验中表现了良好的抗烟草花叶病毒及黄瓜花叶病毒能力．
The bivalent plant expression vector pBICT containing TMV 54KD gene and CMV replicase gene RNA2 with 900bp deletion in it's 31 end was transfered into Agrobacterium tumefaciens strain 311SE. This Agrobacterium was used to transform tobacco by leaf dise method. Regenerated shoots were selected out on MS1 medium with 100μg/ml of kanamycin, 500μg/ml of cefotaxime and rooted on MS0 medium with 25μg/ml of kanamycin,100μg/ml of cefotaxime. Twenty-eight regenerated plants were obtained.The total genomic DNA of 4 regenerated plants and 1 untransformed control plant were extracted and detected by PCR and southern hybridization. The 4 regenerated plants all had TMV and CMV replicase genes in their genomes.The transgenic plants were transplanted to soil and showed replicasemediated resistance to TMV and CMV.
关键词
烟草
复制酶基因
转基因植株
抗病毒植株
TMV
CMV
replicase gene
transgenic plant
Agrobacterium tumefaciens
