Studies On The Introduction Of Hepatitis B Surface Antigen In Lettuce (Lactuca Sativa)

In this experiment, HBsAg (adw subtype) was introduced into lettuce(Lactuca sativa) with the Agrobacterium transformation system and biolistic method. We hope to obtain the transgenic lettue with HBsAg so that it can be used as edible vaccine. The experiment results covered several following points:(1) adw was cloned into nuclear plasmid PB, PBG and chloroplast expression plasmid PLCTB . The expression vectors with adw, namely PB-adw, PBG-adw, PLCTB-adw were constructed by gene recombination technology.(2) For screening the transgenic lettuces from the whole materials, suitable concentration of kanamycin was 100mg/L and spectinomycin was 20mg/L.(3) Regenerated plants with kanamynic resistantance were obtained. PCR , PCR-Southern blot analysis , southern dot blot analysis of lettuce DNA confirmed that adw gene had been integrated into the plant genome. The results also showed that the transformation frequency of PB-adw was higher than that of PBG-adw, which suggested that CaMV35S promoter would be better than PI II promoter in the case of transgenic lettuce.(4) The chloroplast expression plasmid PLCTB-adw, which contained lettuce psb promoter and terminor, besides two homogeneous sequences at adw gene flanks was used to transforme lettuces by the biolislic method. Because selective period of chloroplast transformation was longer than that of the nuclear transformation, only the several weakly regenerated plants with spectinomynic resistantance were obtained. Further screening and molecular detections need to be done.