Expression Of Human Rotavirus Gene In Carrot (Daucus Carota L.var.sativa)

Rotavirus,RV, Reovrridase, Rotaviras are the main pathogens causing diarrhea in children all over the world. Now vaccination is the optical way to control the diarrhea.Oral vaccines produced by transgenic plants would change the traditional means of production and inoculation of vaccines,and the cost of vaccine production would be reduced greatly. Consequently it is very significant to develop transgenic plant vaccines.VP7 and VP4 are major outer capsid protein and they are primary candidates for inclusion in a subunit or recombinant.VP6 is main virus protein, which can induce body to produce antibody. So that introducing VP7,VP4 and VP6 genes into carrot to develop edible plant vaccine of rotaviruses is provided with commercial meaning and applicative prospect.Various factors influencing tissue culture of carrot were investigated and a transformation system was established with explant. Transgenic carrot was obtained by Agrobacterium tumefaciecs-mediated transformation of hypocotyls.VP7,VP6 and VP4 gene was introduced into carrot. The results indicated that the introduced gene was integrated into the T0 generation transgenic plants. The carrot plants of T1 generation integrated different VP7 and VP6 gene were obtained by sexual hybridization .The results of PCR,Western blot and Elisa of T2 transgenic plants showed that the two target gene were expressed in roots of transgenic carrots.The results indicated that:1. Marinated in 1%AgNO3 for 15-20 min was the optimal sterile condition of seeds; 0.1mg/L 2,4-D or 0.1mg/L 2,4-D+0.2mg/L KT was optimal to induct the callus of carrot hypocotyls; MS medium is available for rooting of seedling; 7-9 days is the optimal time to harden regenerated plantlets in bottles best to raise transplanting survivalrate.2. The optimal explants age for transformation was 2-3 days; the Agrobacterium tumefaciens in the logarithm time have the most ability for transformation; the optimal time of transformation was 20 min when the Agrobacterium tumefaciens was diluted to OD600=0.6; the optimal duration of pre-culture was 2-3 days.The experiment about sensitivity of hypocotyls to kanamysin and carbencillin showed that the optimal kanamysin concentration is 80mg/L for transgenic callus selecting,120mg/L for transgenic shoot and regenerated plant selecting; and the suitable carbencillin concentration is 500mg/L.3. Regenerated Kan-resistant carrot plantlets of 70 VP7 gene,68 VP6 gene and 68 VP4 gene were gained and analyzed by PCR, 30%,22% and 21% of the plantlets were confirmed that target gene had been inserted into plant genome; Then the positive plantlets with target gene were further confirmed by Southern blot and RT-PCR that the target gene had been inserted into the carrot genome and transcripted successfully.4. The carrot plants (T1 generation) integrated VP7 and VP6 gene were obtained by sexual hybridization between two group of transgenic carrots which contain one kind of target gene respectively.5. The results of PCR, Western blot and Elisa of T2 transgenic plants showed that the VP7 and VP6 gene were expressed in roots of transgenic carrots.