| Maize is not only the staple food crop, but also an important raw material for feed and industry, nevertheless the threat of pests and weeds leads to a serious decline in its output and quality. The birth of genetic engineering provides an effective means for the prevention and treatment of pests and feeds.G2-epsps gene was cloned from a Psedomonas fluorescens strain G2 isolated from an extremely glyphosate polluted environment, and it was a novel gene which has been patented. In order to improve the glyphosate-tolerant G2-epsps gene's expression level in transgenic maize, the codon of G2-epsps was optimized according to the plant's codon preference, and the synthetic gene was renamed 2mG2-epsps gene. The optimized gene's G+C content was adjusted from the original 58% to 53%, and its nucleotide similarity to G2-epsps gene is 89%。In order to make the 2mG2-epsps gene's encoding product located in chloroplasts correctly, we cloned the 244bp chloroplast transit peptide sequence of Arabidopsis thaliana EPSPS enzyme, and the sequencing results showed that the sequence's similarity with the known sequence is 100%.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. The cry1Ie gene is another Bt insecticidal crystal protein gene with independent intellectual property rights. Its encoding protein has some toxicity not only to the sensitivity Asia corn borer (Ostrinia furnacalis) but also to the resistance Asia corn borer. Cry1Ie has no cross-resistance with Cry1Ab or Cry1Ac protein, thus combining cry1Ah and cry1Ie to the same expression vector can more effectively overcome the problems such as the gene's high homology, single species, pest resistance to Bt protein and so on, at the same time insect-resistant transgenic plants with higher toxicity is expected to be obtained.In this study, insect-resistant cry1Ah gene and glyphosate-toleranct 2mG2-epsps gene was used to construct an insect-resistant/glyphosate-tolerance bivalent plant expression vector, glyphosate isopropylamine salt as a screening agent, 2mG2-epsps gene as a selectable maker gene, the vector was transfer into maize immature embryonic calli by microprojectile bombardment, 80 T0 transformed plants were obtained. To further analyze the T0, T1 and T2 generation plants, the transgenic plants'molecular detection, insect-resistant bioassay and glyphosate-tolerance field test revealed that a total of five transgenic lines inherited to next generation stably, and also had glyphosate-tolerance and resistance to Asia corn strains.A trivalent plant expression vector containing Bt cry1Ah gene, cry1Ie gene and glyphosate-tolerant 2mG2-epsps gene was constructed, glyphosate isopropylamine salt as a screening agent, 2mG2-epsps gene as a selectable maker gene. The vector was transfer into maize immature embryonic calli by microprojectile bombardment, and 69 T0 generation plants were obtained. PCR analysis showed that 17 plants had the integration of insect-resistant cry1Ah, cry1Ie gene and glyphosate-tolerance 2mG2-epsps gene. RT-PCR analysis showed that the foreign gene can be transcript correctly. This study provides research materials for the breeding of multi-gene, dual- resistant transgenic maize.In this study 2mG2-epsps gene was used to be a selection marker gene, and a series of experiments were designed for determining the time of their screening and selection pressure. The results showed that 1 mM glyphosate isopropylamine and 15 days screening can be effectively inhibited non-transgenic maize callus tissue differentiation. So this research work has provided a safe and effective screening system for corn and other monocotyledons crops.
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