| Peanut or groundnut(Arachis hypogaea L.) is an important commercial crop worldwide and provides an excellent source of protein and other nutrients, whose seeds contain about 25% protein. So it is one of the ideal vectors in oral vaccine of transgenic plant.The Agrobacterium strain LBA4404 harboring the binary vector pBI121 was used for transformation. The plasmid carries genes for VP4 (2350bp) of the human rotavirus (HRV) driven by CaMV 35S promoter and neomycin phosphotransferase (NPTⅡ). Two peanut cultivars were used as source of cotyledon explants in this experiment. Cotyledon explants from mature peanut seeds (Arachis hypogaea L.) were optimized to obtain adventitious shoot buds with high frequencies . The percentage of shoots induced of Huayu No.23 and Luhua No.14 can reach to 89.67% and 83.33% respectively in MSC1 with 5.00mg/L 6-BA and 1.50mg/L 2,4-D. The method reported here provides new opportunities for the crop improvement of peanut via genetic transformation.Transformation efficiency could be affected by many factors in the system of cotyledon explants. The better transformation efficiency was obtained by using Agrobacterium tumefaciens strain LBA4404 with the proper OD600 value(0.5-0.7) and with 10-min invasion,3d co-cultivation and the proper concentration of wounded tobacco leaf extract(1.0ml/50ml). The media with 250mg/L Cef and 250-500mg/L Carb could not only lower pollution ratio but also get higher transformation efficiency.Efficient transformation of these cotyledons by using Agrobacterium tumefaciens strain LBA4404 carrying NPTⅡand VP4 gene on binary vectors led to the production of a large percentage of transgenic plants. Transformed individuals were obtained through selection on medium containing 125 mg /L Kan. Integration of the transgenes were assessed by PCR amplification and Southern blot hybridizations. Taking pBG1VP4 or pBG1VP4 plasmid as positive control, non-transformed peanut as negative, 22 plants among 26 plants which can grow up through selection on medium containing 125mg/L Kan were assessed by PCR amplification of 620-bp fragment of NPTⅡgene. Then all of 22 plants were assessed by PCR amplification of 2350-bp fragment of VP4 gene.Taking VP4 gene withα-32P-dCTP maker as probe, 5 plants selected radomly from 22 positive plants were analysed by PCR-Southern blot hybridizations and all of them have DNA bloting bands. Then the genomic DNA of 4 plants chosen from PCR-Southern positive plants were further analyzed with Southern blot hybridizations and all have correspondent DNA bloting bands. The results showed that the foreign gene has been integrated into genome of transformated peanuts. The totle RNA from 11 plants of Luhua No. 14 has been assessed by RT-PCR analysis and the result show the expression of G1VP4 gene.An edible vaccine based on the VP4 of human rotavirus (HRV) could provide a means to protect children from severe acute diarrhea, which can elicit humoral and cell-mediated responses of the mucosal and systemic immune systems, evoke less pain and discomfort than parenteral delivery, and eliminate needle associated risks. Transgenic peanuts are an ideal means by which to produce oral vaccines, as the rigid walls of the plant cell protect antigenic proteins from the acidic environment of the stomach, enabling intact antigen to reach the gut associated lymphoid tissue. Elevated expression of the rotavirus VP4 antigen in transgenic peanuts is a critical factor in the development of a safe and effective rotavirus vaccine. Our increasing ability to manipulate the immune system offers hope that, in the future, at least some of these infections may be prevented by edible vaccine.
|
PDF Full Text Request