Study On Insect-resistant Transgenic Maize (Zea Mays L.) Harboring Bt Cry1 Ah Gene

The cry1Ah gene was one of the novel insecticidal genes cloned from Bacillus thuringiensis isolate BT8 which exhibited high toxicity against lepidopteran larvae. Cry1Ah protein showed higher toxicity to Asian corn borer (Ostrinia furnacalis) than any other cry1A genes. Transferring cry1Ah gene into maize can not only improve the resistance of transgenic maize to Asian corn borer but also delay the insect resistance risk. The cry1Ah gene was a candidate gene for insect resistant transgenic maize research.To confirm the cry1Ah gene's function in transgenic maize, a plant expression vector pHUAh harboring the cry1Ah gene was constructed and transferred into immature embryonic calli of maize Q31×Z31 by microprojectile bombardment. The hyg gene was used as the selective marker gene. Sixty-six regenerated plants were obtained in T0 generation,thirteen of which were PCR positive. Bioassay results of T1 transgenic plants showed that events B1 and B7 were high resistant to the Asian corn borer . The two events were monitored by molecular detection and bioassay for 4 years from T1 toT5 generations. The results showed that foreign gene cry1Ah was expressed stably in maize and could be inherited stably over generations. Results of bioassay to T2-T5 transgenic maize plants suggested that the transgenic plants had high resistance against the Asian corn borer .A marker-free plant expression vector pUAh harboring the cry1Ah gene was constructed and transferred into maize inbred line X090 by pollen-tube pathway. Two thousands and two hundreds regenerated plants were obtained in T1 generation and were detected for the expression of Bt toxic Cry1Ah protein by Jin-biao immune detection strips. There were 172 plants expressing the Cry1Ah protein,35 of which were insect resistant confirmed through bioassay. In T2 generation, there were 12 transgenic events showing high resistance against the Asian corn borer which were further analyzed by DNA assay, protein assay and bioassay from T2 to T5 generations. Results of DNA analysis from T2 to T5 generations showed that foreign gene cry1Ah could be inherited stably over generations in transgenic maize. Nine homozygous transgenic events were obtained in T5 generation which showed uniformly positive in PCR assay. Protein analysis suggested that the Cry1Ah protein were stably expressed in each generation in transgenic maize and the contents of Cry1Ah protein were different among the 12 transgenic events as well as among different tissues. Content of Cry1Ah protein was the highest in transgenic event P12 ( line of P12-8-7-13 ) which was 0.130% of the total soluble protein . The lowest was the event P16(line of P16-16-6-5),of which the content of Cry1Ah protein was 0.023%. Detection of Cry1Ah protein in different tissues of event P12 showed that the Cry1Ah content was the highest in the husk leaf which was 0.186% of the total soluble protein. Bioassay results exhibited that the transgenic maize harboring cry1Ah gene showed high resistance against the Asian corn borer and the insect resistance could be inherited stably which were positively correlated with the level of the Cry1Ah expression. In this study, the integration 3'flanking sequence of cry1Ah gene in event P19 was analyzed by TAIL-PCR. The results showed that the flanking sequence was the highest homologous with the sequence at the BIN4.09 locus in the maize genome. The identity of nucleotide sequence was 99.5% .These revealed that foreign gene had been integrated into the maize genome.The stably inherited transgenic maize obtained in this study which had no selective marker gene could be potential candidates for the breeding of Bt insect-resistant transgenic maize.