转抗虫基因三倍体毛白杨植株体内农杆菌残存与逃逸

用部分改造的BtCry1Ac基因与慈菇蛋白酶抑制剂（APLA）基因构建的双抗虫基因表达载体，通过农杆菌介导法对三倍体毛白杨进行了转化，对转化后植株体内残存农杆菌在继代培养和移栽过程中进行了跟踪检测。结果表明：通过对转化再生植株的分子生物学检测，42个株系中，33个株系为阳性，阳性率达到80％；用Bt毒蛋白抗血清进行ELSA检测结果表明，7个转基因株系都有Bt杀虫蛋白表达；基因转化后，可采用附加50mg／L卡那霉素，300mg／L羧苄青霉素的筛选培养基消除细菌并进行抗性芽筛选。对28个转基因株系叶片、茎段和根段在含有卡那霉素50mg／LYEB培养基上进行细菌培养，通过在T-DNA区、质粒Vir区和农杆菌基因组设计引物，进行PCR检测，证明有3个株系（33、37、5号）检测到残存工程农杆菌，并在组培瓶中存活24个月。将带菌的3个株系组培苗移栽到花盆中，室内培养1个月后，在33号株系根际土壤中检测到了目的农杆菌。

Survival and escape of Agrobacterium tumefaciens in triploid hybrid lines of Chinese white poplar transformed with two insect-resistant genes

Two partly modified insect-resistant genes (BtCryI Ac gene Bt gene toxin against Lepidopterean insects] and API gene arrowhead proteinase inhibitor]) were transferred to the triploid hybrid of Chinese white poplar ((Populus tomentosa Carr. × Populus bolleana Louche) × Populus tomentosa Carr.) mediated by A. tumefaciens. The survival of Agrobacterium in transgenic plants was examined during the processes of transplanting and subculturing on the nutrient medium. The results suggested that 80% of the plants, which were obtained by repeated selection on media added with 50 mg/L kanamycin and 300 mg/l carbenicillin, showed positive reactions after examination using molecular methods. The ELISA test indicated that the Bt toxoprotein was expressed in seven of the transgenic sub-clones. Leaves, stems, and roots of all the 28 transgenic plants were cultured on the YEB medium added with 50 mg/L kanamycin, and it was found that Agrobacterium survived in three sub-clones (33, 37, 5) and could have existed for 24 months in the bottle. These three transgenic sub-clones were transplanted and cultivated for one month in the room, and then the target Agrobacterium was found in rhizosphere of the sub-clone 33.