To increase the expression efficiency of the Bt gene in transgenic poplar and expand the insect resistance spectrum, Cry1Ac and Cry3A were simultaneously constructed in two different plant transformation vectors. Two Bt genes on vector p71A68Y71 were separately driven by promoters CAMV35S and CoYMV, and a matrix attachment region (MAR) sequence was added to both sides; in contrast, two Bt genes on vector p05A68A71 were driven by the promoter CAMV35S without a MAR sequence structure. With the agrobacterium mediation method, two vectors transformed the high-quality hybrid clone poplar Juba of Populus deltoides (W. Bartram ex Marshall) and thus produced four transgenic resistant lines. Polymerase chain reaction (PCR) showed that the targeted genes were integrated into the poplar Juba genome. Fluorescence quantitative PCR and enzymelinked immunosorbent assay techniques were used to detect the transcription abundance and toxic protein expression of the Bt gene in the leaves of eight transgenic leaves. Results show the significant difference in transcription abundance of the two Bt genes between different lines. The transcription abundance of the gene Cry1Ac of the K series lines from vector p71A68Y71 was significantly higher than that of the H series lines from the other vector; however, no significant difference in the transcription abundance of gene Cry3A between the two vectors was observed. The transcription abundance of gene Cry3A was higher than that of gene Cry1Ac. Significant differences in the toxic expression of the two Bt genes existed among the lines but not in the H and K series lines. The Cry3A toxic protein content was much higher than the Cry1Ac toxic protein content in all lines, and the expression of the latter at 0.13 ng·g −1 to 0.69 ng·g −1 was extremely low. The insect-resistant effect of the transgenic lines to lepidoptera pests such as the Hyphantria cunea (Drury) larva was unremarkable, and no significant difference was found in different lines and vectors. The lines strongly resisted coleoptera pests, e.g., Plagiodera versicolora (Laicharting), and their insectresistant effect was significantly higher than that of the highly resistant trans-Cry3A poplar line CC84. Significant differences were found between different lines, but no significant difference was found between the K and H series lines, which were from different vectors. Trangenic lines H4 and K1 had a higher transcription abundance, toxic protein expression, and lethal effect on P. versicolora than the other lines.Résumé : Afin d'augmenter l'efficacité du gène Bt chez le peuplier transgénique et d'élargir le spectre de résistance à l'insecte, les gènes Cry1Ac et Cry3A ont été insérés simultanément dans deux vecteurs de transformation de plante. Les deux gènes Bt sur le vecteur p71A68Y71 étaient contrôlés séparément par les promoteurs CAMV35S et CoYMV, et une séquence de région d'attache matricielle (MAR) a été ajoutée de chaque côté. De façon différente, les deux gènes Bt sur le vecteur p05A68A71 étaient contrôlés par CAMV35S sans st...