A novel insecticidal gene crylAh was cloned from Bacillus thuringiensis isolate BT8 previously for plant genetic engineering improvement. Truncated active CrylAh toxin has a toxicity level similar to that of the full-...A novel insecticidal gene crylAh was cloned from Bacillus thuringiensis isolate BT8 previously for plant genetic engineering improvement. Truncated active CrylAh toxin has a toxicity level similar to that of the full-length CrylAh toxin. In this study, plant expression vector pMhGM harboring truncated crylAh gene was transformed into maize （Zea mays L.） immature embryos by Agrobacterium tumefaciens-mediated transformation at which maize alcohol dehydrogenase matrix attachment regions （madMARs） were incorporated on both sides of the gene expression cassette to improve gene expression. A total of 23 PCR positive events were obtained with a transformation efficiency of 5% around. Bioassay results showed that events 1-4 and 1-5 exhibited enhanced resistance to the Asian corn borer （Ostriniafurnacalis）. These two events were further confirmed by molecular analysis. Southern blot suggested that a single copy of the crylAh gene was successfully integrated into the maize genome. Western blot and ELISA showed that the foreign gene crylAh was expressed stably at high level in maize and could be inherited stably over generations. The results of a bioassay of T l-T4 transgenic maize plants indicated that the transgenic plants were highly toxic to the Asian corn borer and their resistance could be inherited stably from generation to generation. Thus, events 1-4 and 1-5 are good candidates for the breeding of insect-resistant maize.展开更多
基金the financial support of the Genetically Modified Organisms Breeding Major Project, China (2011ZX08003-001)the National Basic Research Program of China (973 Program, 2009CB118902)the National Natural Science Foundation of China (30970231)
文摘A novel insecticidal gene crylAh was cloned from Bacillus thuringiensis isolate BT8 previously for plant genetic engineering improvement. Truncated active CrylAh toxin has a toxicity level similar to that of the full-length CrylAh toxin. In this study, plant expression vector pMhGM harboring truncated crylAh gene was transformed into maize （Zea mays L.） immature embryos by Agrobacterium tumefaciens-mediated transformation at which maize alcohol dehydrogenase matrix attachment regions （madMARs） were incorporated on both sides of the gene expression cassette to improve gene expression. A total of 23 PCR positive events were obtained with a transformation efficiency of 5% around. Bioassay results showed that events 1-4 and 1-5 exhibited enhanced resistance to the Asian corn borer （Ostriniafurnacalis）. These two events were further confirmed by molecular analysis. Southern blot suggested that a single copy of the crylAh gene was successfully integrated into the maize genome. Western blot and ELISA showed that the foreign gene crylAh was expressed stably at high level in maize and could be inherited stably over generations. The results of a bioassay of T l-T4 transgenic maize plants indicated that the transgenic plants were highly toxic to the Asian corn borer and their resistance could be inherited stably from generation to generation. Thus, events 1-4 and 1-5 are good candidates for the breeding of insect-resistant maize.
