van Wordragen M F, Honée G, Dons H J
Department of Developmental Biology, DLO-Centre for Plant Breeding and Reproduction Research (CPRO-DLO), Wageningen, Netherlands.
Transgenic Res. 1993 May;2(3):170-80. doi: 10.1007/BF01972611.
A 3'-end truncated crystal protein gene, derived from Bacillus thuringiensis (Bt) subsp. aizawai 7.21, encoding the toxic fragment of the insecticidal protein cryIA(b), was constructed. The gene was inserted into a transformation vector, also carrying the neomycin phosphotransferase II (nptII) gene and the beta-glucuronidase (gus) gene, and introduced in the oncogenic Agrobacterium tumefaciens strain A281, harbouring the Ti-plasmid pTiBO542. The recombinant Agrobacterium strain was used to transform leaf explants of chrysanthemum (Dendranthema grandiflora) cultivar Parliament. The resulting tumours were kanamycin-resistant, exhibited beta-glucuronidase activity and produced agropine and mannopine. In most tumours, all simultaneously transferred genes were expressed, owing to selection for the presence of both T-DNAs, but no correlation was found between the level of expression of the various genes. A bioassay was developed, in which larvae were fed with tumorous chrysanthemum tissue, in order to detect the effect of the transferred toxin gene on larval development. Using this bioassay with second instar larvae of Heliothis virescens (tobacco budworm), 17 tumour lines were tested. Several of these lines proved to be strongly inhibitory to larval growth. These results indicate that Bt-based insect resistance might be used as a tool in reducing the amount of pesticides used in chrysanthemum culture.
构建了一个源自苏云金芽孢杆菌（Bt）日本亚种7.21的3'端截短的晶体蛋白基因，该基因编码杀虫蛋白cryIA(b)的毒性片段。将该基因插入到一个转化载体中，该载体还携带新霉素磷酸转移酶II（nptII）基因和β-葡萄糖醛酸酶（gus）基因，并导入携带Ti质粒pTiBO542的致癌根癌土壤杆菌菌株A281中。用重组土壤杆菌菌株转化菊花（Dendranthema grandiflora）品种Parliament的叶片外植体。产生的肿瘤对卡那霉素具有抗性，表现出β-葡萄糖醛酸酶活性，并产生农杆碱和甘露碱。在大多数肿瘤中，由于对两个T-DNA的存在进行选择，所有同时转移的基因都得以表达，但未发现各基因表达水平之间存在相关性。开发了一种生物测定法，用肿瘤菊花组织喂养幼虫，以检测转移的毒素基因对幼虫发育的影响。使用这种生物测定法对烟芽夜蛾（Heliothis virescens）的二龄幼虫进行检测，测试了17个肿瘤系。其中几个系被证明对幼虫生长有强烈抑制作用。这些结果表明，基于Bt的抗虫性可作为减少菊花栽培中农药用量的一种手段。
