Agrobacterium-mediated Insect-Resistant Transgenic Maize Harboring Bt Cry1Ah Gene

At present some transgenes for plant genetic engineering were from microbiology. The codon of microbiology genes′coding sequence was mostly plant rare codons, which could lead to genes'expression very low in plants. Codon optimization could significantly improve foreign genes'expression in plants.Bt cry1Ah gene was a novel insecticidal protein gene cloned from a Bacillus thuringiensis isolate BT8 in China, and its encoding product′s insecticidal activity to lepidopterous pests was higher than the currently used cry1Ac or cry1Ab. Previous work had done that the cry1Ah gene was modified according to the plant codon bias in our laboratory.In this study, the first modification of cry1Ah gene and glyphosate-toleranct 2mG2-epsps gene were used to construct an insect-resistant/glyphosate-tolerance bivalent plant expression vector pAhmG, glyphosate isopropylamine salt as a screening agent, 2mG2-epsps gene as a selectable maker gene, the vector was transfer into maize inbred Z31 immature zygotic embryos by Agrobacterium-mediated. Forty transformed plants of T0 generation were obtained, fourteen of which were positive events through molecular detection. To further analyze the T1 generation plants, the transgenic plants′molecular detection and insect-resistant bioassay were done. The transgenic plants′molecular detection including PCR,RT-PCR,Western blot,ELISA revealed that Bt cry1Ah gene was expressed and inherited to next generation stably. But in this study, the expression of Cry1Ah protein in transgenic maize was found very low (the highest is 0.008%) and degradation that reduced the effect of insect resistance.Then, according to the maize coden bias, the cry1Ah gene was modified again. The second modified cry1Ah gene′s nucleotide similarity to the first modified cry1Ah gene was 75.95%. The two plant expression vectors pmAhb and pmAhIeb were constructed, bar gene as a selectable maker gene. The vectors were transfered into maize inbred Z31 immature zygotic embryos by Agrobacterium-mediated transformation, obtaining thirty-three transformed plants of T0 generation. Through PCR and RT-PCR detections, fourteen positive transgenic events were obtained. Western blot and ELISA detections were done. The Cry1Ah protein was correctly expressed in transgenic maize and the highest expression was up to 0.045% of maize soluable proteins, which were 5-10 times higher than the expression in transgenic plants harboring pAhmG vector.In this study, the expression of cry1Ah gene was improved through codon optimization, and the transgenic maizes with Asian corn borer resistance were obtained. It laid the foundation for the efficient use of foreign genes and the development of insect-resistant transgenic maize in the future.