Establishment Of Efficient Regeneration System Of Carrot Hypocotyls And It's Transformation With Rotavirus Related Protein Genes

Rotavirus(RV) infection is a major cause of dehydrating diarrhea in children all over the world, especially in developing country. 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, and can induce body to produce antibody. So introducing vp7,vp4 and vp6 genes into carrot to develop edible plant vaccine of rotaviruses would change the traditional means of production of vaccines and the cost of vaccine production would be reduced greatly. It is provided with commercial meaning and applicative prospect.In this study, vp4,vp7 and vp6 genes were introduced into carrot and high-efficient regeneration system and genetic transformation system of carrot were established.Effects of several factors on the regeneration of carrot hypocotyls and genetic transformation of carrot mediated by Agrobacterium tumefaciens were studied. The results indicated that: ① Marinated in 1%AgNO3 for 15～20 min was the optimal sterile condition of seeds; ② 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; ③ MS medium is available for rooting of seedling; ④ 7～9 days is the optimal time to harden regenerated plantlets in bottles best to raise transplanting survivalrate; ⑤ The experiment about sensitivity of hypocotyls to kanamysin and carbencillin showed that the optimal kanamysin concentration is 75mg/L for transgenic callus selecting , 120mg/L for transgenic shoot and regenerated plant selecting; and the suitable carbencillin concentration is 500mg/L. ⑥ The optimal co-culture time was 2～3 days.Finally, hundreds of regenerated Kan-resistant carrot plantlets were gained and analyzed by PCR, 62 of 204 plantlets were confirmed that the vp7 gene had been inserted into plant genome; 33 of 120 plantlets were confirmed that the vp6 gene had been inserted into plant genome; 21 of 94 plantlets were confirmed that the vp4 gene had been inserted into plant genome. Then plantlets with vp7 gene were further confirmed by Southern blot and RT-PCR that the target gene had been inserted into the carrot genome and transcripted successfully.