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苏云金杆菌HD-1的杀虫蛋白基因经5′端改造,3′端进行4种不同长度缺失后插入到含有双增强子的35S启动子,翻译增强子“Ω′”片段的双元载体中,借助土壤农杆菌LBA 4404将在此双元载体上的杀虫蛋白基因及新霉素磷酸转移酶基因(NPTII)转入到生产品种烟草NC89的染色体上,从而获得了抗卡那霉素的转化再生烟草植株。用1—3龄烟青虫对这些转化植株进行大量重复虫试结果表明用4种不同长度B.t.基因转化的再生植株中都有抗虫性高的植株,其中以1.8kb的B.t.Cry IA(c)基因转化的植株杀虫效果最好,这一组转基因植株的平均杀虫率在90—100％的约占该组总虫试植株的50％。对高抗虫性植株的子一代(T1)和子二代(T2)进行遗传分析,分子生物学分析和进一步的抗虫试验表明B.t.基因已遗传到子代并初步选到了高抗虫性的转基因纯合株系D8-14和D19-8等。
The insecticidal protein gene of Bacillus thuringiensis HD-1 was transformed at the 5 ’end and inserted into the binary vector containing the 35S promoter of the double enhancer and the “Ω” fragment of the translation enhancer after being subjected to 4 different length deletions at the 3’ end , The insecticidal protein gene and the neomycin phosphotransferase gene (NPTII) on the binary vector were transferred into the chromosome of the production variety Tobacco NC89 by means of the Agrobacterium tumefaciens LBA 4404 to obtain the anti-kanamycin-resistant Transformation of regenerated tobacco plants. A large number of replica tests on these transformed plants with 1-3-year-old tobacco budworm showed that plants regenerated from 4 regenerated Bt genes of different lengths all had highly insect-resistant plants, with 1.8 kb of BtCry IA (c) The transgenic plants had the best insecticidal effect. The average insecticidal rate of the transgenic plants in this group was about 90% to about 50% of the total plant test of the group. Genetic analysis, molecular biology analysis and further insect-resistant tests on the first generation (T1) and second generation (T2) of the plants with high resistance to insects showed that the Bt gene has been inherited into progenies and the transgenic plants with high insect resistance have been preliminarily selected Homozygous lines D8-14 and D19-8 and so on.