Expression Of The Modified Cry1Ac,crylIe Genes In E.coli, Transgenic Tobacco Plants And Transgenic Maize Plants

The coding regions of wild Bacillus thuringiensis cry1Ie and cry1Ac gene were modified according to plant preferring codons. The modified crylle and crylAc gene were artificially synthesized. Modified crylle and crylAc gene were cloned into prokaryotic expression vector pET28b, to construct plasmid pETIe and pETAc. These two proteins' expression in E. coli BL21(DE3) were confirmed by SDS-PAGE analysis. Proteins purified were injected into rabbit to prepare polyclonal antibodies. Bioassays using crude expression products in E. coli revealed that Cry1Ie and Cry1Ac proteins had a similar toxicity to corn borer as wild type proteins.Modified cry1Ac and cry1Ie gene had also been cloned into plant expression vector p3301, to construct plasmid p3301ubiAc and p3301ubiIe in which two genes were under the control of maize ubiquitin-1 promoter respectively. Tobacco plant leaves were transformed with these two plasmids and transgenic tobacco plants were obtained. The results of PCR and Sourthern blot showed that crylAc and crylle gene have integrated into tobacco plants genome. The results of insect assay with Asian corn borer showed that transgenic tobacco plants carrying crylAc and crylle showed insecticidal activity against corn borer.Potato proteinase inhibitor II (pinll) signal peptide sequence was obtained by the method of PCR. To allow secretion of the CrylAc protein into the intercellular space, potato proteinase inhibitor II (pinll) signal peptide sequence was N-terminally fused to the crylAc coding region to construct plasmid p3301ubisigAc. Expression of CrylAc in transgenic tobacco plants was assayed with ELISA. The results showed that pinll signal peptide sequence enhanced the expression of CrylAc protein in transgenic tobacco. GFP gene was also fused behind the signal peptide sequence to construct plasmid p3301ubisigGFP and transformed to tobacco. The results of fluorescent detection showed GFP localization in the apoplast.The promoters of maize chlorophyll a/b binding protein (cab promoter) and maize extension protein gene (silk promoter) were cloned by the methods of PCR. These two promoters, maize ubiquitin promoter and crylle, crylAc gene were used to construct plant expression plasmid p3301cabIeubiAc, p3301silkIe and p3301ubiAc. These plasmids were used to transform maize Primary embryogenic calli and transgenic maize plants were obtained. The results of evaluating of the resistance to Asian corn borers showedsome Bt transgenic maize lines showed good insect-resistance.