Using linker peptide LP4/2A for multiple gene transformation is considered to be an effective method to stack or pyramid several traits in plants. Bacillus thuringiensis（Bt） cry gene and epsps（5-enolpyruvylshikimate-3-phosphate synthase） gene are two important genes for culturing pest-resistant and glyphosate-tolerant crops. We used linker peptide LP4/2A to connect the Bt cry1 Ah gene with the 2m G2-epsps gene and combined the wide-used man A gene as a selective marker to construct one coordinated expression vector called p2 EPUHLAGN. The expression vector was transferred into maize by Agrobacterium tumefaciens-mediated transformation, and 60 plants were obtained, 40% of which were positive transformants. Molecular detection demonstrated that the two genes in the fusion vector were expressed simultaneously and spliced correctly in translation processing; meanwhile bioassay detection proved the transgenic maize had preferable pest resistance and glyphosate tolerance. Therefore, linker peptide LP4/2A provided a simple and reliable strategy for producing gene stacking in maize and the result showed that the fusion gene transformation system of LP4/2A was feasible in monocot plants.
SUN HeLANG Zhi-hongLU WeiZHANG JieHE Kang-laiZHU LiLIN MinHUANG Da-fang
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.
LI Xiu-yingLANG Zhi-hongZHANG JieHE Kang-laiZHU LiHUANG Da-fang
