| Soybean(Glycine max(L.)Merrill.)is an important crops of oil and food.It has been regard as a valuable source of protein for human nutrition.However,soybean can be suffered from soybean pod borer(Leguminivora glycinivorella(Mats.)),Soybean pod borer which belongs to Lepidoptera,Olethreutidae,is one of the main pod moth pests.The larvae of soybean pod borer eat the pods and grain,speacially.The results decrease the yield and quality of soybean.The traditional strategy of prevention and treatment of soybean pod borer is the spraying of chemical pesticides,biological control and conventional breeding.A mount of pesticide has been used for preventing the damage caused by soybean pod borer,which not only causes healthy problem in humans and animals,but imbalances the wild environment,such as the pesticide residue in food and for increased resistance of pathogen.But,natural bio-insecticides haven't got general application due to its high cost,instability in field trial and narrow anti-insect spectrum.Because of the deficiency of gene source,and long breeding period of traditional breeding methods,traditional methods cannot satisfy the demand for selecting new type to resist pest in agriculture.The Plant Genetic Engineering can overcome the difficulty in distant hybridization,decrease the breeding period and improve the type major agronomic traits.Applying of Plant Genetic Engineering to make plant expressing the Insect-resistant gene has been regard as the significant method to prevent pest and a way to breeding new type lines.Vectors were constructed by the modified cry2Aa9m gene,which has resistance to soybean pod borer,and herbicide resistant gene "bar",expressing by pod-specific expression promoter or seed-specific expression promoter respectively.The transformation of cry2Aa9m gene into soybean by Agrobacterium Tumefaciens,Transgenic plants were obtained,and initial insect resistant assay was made by artificial infestation.They could be used as the gene source of breeding borer-resistance varieties.And we established an effective system of soybean regeneration and genetic transformation.The main results were summarized as follows:1.The construction of plant expression vectorsTwo plant expression vectors were constructed.Within the vectors of pCMB2A and pCGB2A,the modified cry2Aa9m gene expression is driven by pod-specific promoter Pmsg and seed-specific promoter PGy2,respectively.The selectable maker is bar gene,and the modified cry2Aa9 gene expression is included.2.The transformation mediated by Agrobacterium TumefaciensThe concentration of Glufasinate was identified as 1.5 mg/L and 3.0 mg/L on stage of shoot induction of Suinong 28,Hefeng50.The transgenic acceptors were the cotyledonary node explants.cry2Aa9m gene,which were respectively regulated by tissue specific promoter Pgy2 and Pmsg,was transferred into soybean,including bar gene.And we obtained the massive resistant plants.3.The identification of resistant plantsUsing the method of PCR and RT-PCR to analysis gene integration of T0?T1 generation,2 of T0 and 1 of T1 pCGB2A with Glufasinate resistant transgenic plants,6 of T0 and 4 of T1 pCMB2A with Glufasinate resistant transgenic plants,1 of T0 and 4 of T1 pCMBA with Glufasinate resistant transgenic plants were obtained.4.Insect resistant assay of transgenic resistant plantsImmature pods of 15 PCR positive plants were treated by insect resistant assay which was made by artificial infestation.Result suggested that three transgenic plants had high susceptibility,five had susceptibility,and another three had mid susceptibility. |
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