| Maize is an important feed, industrial raw material and food crop, the global planting quantity is just below of rice and wheat. Corn has important significance on the national economy of our country, which has become one of the main crops of northern region, along with economic development and population increasement, the demand of corn in our country increases continuously. While the corn borer has been main pest of maize, it cause heavy loss in the production. Each year the damage loss due to insects accounted for about more than15%of the crop yield. Corn borer may damage every part of corn plant above ground, so that the victim parts lose functions and grain yield may be reduced. Traditional breeding methods led to slow progress of the breeding because of its long time breeding and lack of breeding germplasm resources. Gene engineering breeding has attracted more and more attentions of researchers in various countries. Bt is the abbreviation of Bacillus thuringiensis, which is a gram positive bacillus. Bt insecticidal protein produced by Bacillus thuringiensis can destroy the insect gut cell osmotic pressure, resulting in the death of insect. While the Bt gene originates from bacterial gene, its wild-type gene is not suitable to express in higher plants. From the research results, the wild type gene can be transformed appropriately so as to improve its expression quantity in higher plants.The coding regions of wild Bacillus thuringiensis Cry1Ab gene was modified according to plant preferring codons. The modified CrylAb gene were arificially synthesized. Modified CrylAb gene was cloned into prokaryotic expression vector pET28b, to construct plasmid pETAb. The protein'expression in E.coli BL21(DE3) was confimed by SDS-PAGE analysis and immune test paper. The results show that most of protein is inclusion body, but there is still a certain amount of soluble protein. Bioassays using soluble protein in E.coli revealed that Cry1Ab protein had a similar toxicity to corn borer as wild type proteins.Modified Cry1Ab gene hand also been cloned into plant expression vector pCPB, to construct plasmid pCPBAb in which the gene was under the control of CaMV35S promoter. CrylAb gene was introdued into immature embryo callus of PA×PB using particle bombardment. After selection, regeneration culture,53transgenic plants were regenerated.In the process of seedling detection, Some of them were positive by PCR and immune test paper analysis, there were33transgenic plants showed PCR positive. But CrylAb proteins did not all expression in PCR positive plants, there were29transgenic plants showed CrylAb protein expressed. CrylAb protein expression amount is not same, transgenic maizes as independent transformation events. The results of PCR and immune test paper showed that Cry1Ab gene have integrated into maize genome. The results of insect assay with Asian corn borer showed that transgenic maize and non-transgenic maize has significant difference in blade wormhole. There were small and less wormholes in transgenic maize blade, blade wormholes of non-transgenic connected togather and formed a strip-shaped hollowed-out insect speckle.Transgenic maize plants showed insecticidal activity against corn borer.In this study, the wild-type CrylAb gene was reformed successfully, maize immature embryo callus were transformed with Cry1Ab gene and transgenic maize plants were obtained. The results of evaluating of the resistance to Asian corn borers showed some transgenic maize lines showed good insect-resistance. |
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