双抗(抗病毒及抗虫)植物表达载体的构建及番茄的转化鉴定

CONSTRUCTION OF PLANT EXPRESSION VECTOR WITH DOUBLE RESISTANCE TO VIRUS AND INSECT AND IDENTIFICATION OF TRANSFORMATION IN TOMATO

将抗病毒的ＣＭＶ－ｃｐ 基因和抗虫的Ｂｔ－ｔｏｘｉｎ 基因依次插入到植物表达载体ｐＥ３ 的ＨｉｎｄⅢ和ＫｐｎⅠ位点，通过菌落原位杂交筛选和酶切鉴定，然后以土壤农杆菌ＧＶ３１１－ＳＥ介导转化番茄，胭脂碱检测，染色体ＤＮＡ 的点杂交及ＰＣＲ扩增证明ＣＭＶ－ｃｐ 基因和Ｂｔ－ｔｏｘｉｎ 基因已同时导入转化再生的番茄植株。ＲＮＡ 点杂交证明ＣＭＶ－ｃｐ 基因和Ｂｔ－ｔｏｘｉｎ 基因已在转基因番茄植株中同时获得表达。

By in situ hybridization of bacterium clone and analysis of restriction enzyme digestion, both CMV cp gene and Bt toxin gene were inserted one by one into T DNA of binary plant expression vector pE 3 .The reconstructed plasmid was named pE 14 .Then, tomato was transformed with pE 14 mediated by Agrobacterium tumefaciens GV311 SE, four regenerated tomato plants were obtained on the MS medium containing 100 μg/mL kanamycin. Assay of nopaline, dot blotting of tomato genomic DNA and PCR amplication of CMV cp gene and Bt toxin gene from genomic DNA showed that CMV cp gene and Bt toxin gene were transferred into the four regenerated tomato plants simultaneously with T DNA, and no recombination of genes occurred. RNA dot blotting showed that two of them could express simultaneously the CMV cp gene and Bt toxin gene proteins. The resistances to virus and insect of the transgenic tomato plants will be tested in their F 1 and F 2 regenerations.