Study On Genetic Transformation Of Antigen G3VP7 Gene Of Human Rotavirus In Peanut

Peanut has high nutritious value, and also is a low-cost source of protein, containing about 30% protein. Thus it is one of the ideal vector in oral vaccine of transgenic plant.. In order to increase the expression level of target gene and make it easier to purify recombimant protein. The plant binary expression vector is pBOG3VP7 harboring fusion gene oleosin-vp7, which is promoted by ole-promoter. The recombined pBOG3VP7 plasmid was singled out and then transformed into Agrobacterium LBA4404. The single Agrobacterium colonies growing on YEB solid medium (Kan 50mg/L, Str 50mg/L) were detected with polymerase chain reaction (PCR). The colony of LBA4404 containing pBOG3VP7 was used for thransformation. Cotyledon nodes were used as transformation recipients. Genetic transformation system of peanut nodes via Agrobacterium tumefaciens- mediated was developed, in which the effects of several factors influencing the tranformation were investigated and optimized. Peanut nodes were infected with Agrobacterium tumefaciens LBA4404 harboring pBOG3VP7 under the treatment of negative pressure for 10min, co-cultured for 3 days, and regenerative shoots were screened on selective medium after sub-culture of 2 weeks. Concentration of AS with 100μmol/L was helpful for successful infection. Taking pBOG3VP7 plasmid as positive control, non-transformed peanut as negative, vp7 gene with DIG-dATP maker as probe, 6 plants among 11 plants which can grow up through selection on medium containing 125mgl-1 Kanamycin were assessed by PCR amplification of 978-bp fragment of vp7 gene and 3 plants correspondent DNA blotting band by PCR-Southern blot hybridizations, that is, the result is positive. Then the genomic DNA of PCR-Southern positive plants were further analyzed with Southern blot hybridizations. 3 plants had all correspondent DNA blotting band. The results showed that the foreign gene has been integrated into genome of transformated peanuts.