| This research was to grope for the possibility of expression of hepatitis B surface antigen gene in plants.ln order to settle base for facture hepatitis vaccine.The studies on the tissue culture system of 11 kinds of tomatos. Through different concentrations grads combination experiment of 6BA and IAA were carried out, in order to establish optimum culture medium. The component to induce callus of cotyledon of lichun tomato is: MS+6BA1.0mg/H-IAA0.2mg/I; Differentiation regeneration optimum culture medium component is: MS+6BA1.0mg/l+IAA0.02mg/l; Rootage optimum culture medium component of tissue culture seedling is. 1/2MS+IBA1.0mg/l. Thus we had established the optimize tissue culture system of lichun tomato. Excellent lichun tomato was selected as transgnic acceptor. Through Kan allergy mensurate of cotyledon callus to establish filtration concentration is 50mg/l, filtration concentration of rootage is 40mg/L.We had GFP gene whose span is 717bp cloned to the plasmid pB1121. The plasmid pBI121 carrying GFP gene under control of the cauliflower mosaic virus 35S promoter was constructed and transformed into Agrobactrium tumefaciens LBA4404 by the freeze-thaw method. Tomato explants from cotyledon were cocultivated with Agrobactrium tumefaciens LBA4404 with the plasmid pBI121GFP. Inoculated explants were cultured successively on beforehand media cocultive media, selective media and root growth media to establish the optimize heredity transform system, 13 Km-resistant tomato plants were obtained .Distill DNA of the Km-resistant plants, then use the approaches of PCR to identify the selected resistant plants. The aimed gene of Km-resistant tomato plans were enpanded by PCR. Used Digoxigeninin labeled GFP gene as a probe,3 masculine plants 'were analyzed by PCR-Southern blotting, hybridization signals were observed,all these result prove aimed gene have been conformitied into genome of tomato.Through the same way, we had s gene whose span is 681bp cloned to the plasmid pBln438. The plasmid pBIn438S carrying S gene under control of the DE35S promoter was constructed and transformed into Agrobactrium tumefaciens EHA105 by the freeze-thaw method. Tomato explants from cotyledon were cocultivated with Agrobactrium tumefaciens EHA105 with the plasmid pBIn438S. Based on the optimize heredity transform system, 3 Km-resistant tomato plants were obtained .Distill DNA of the Km-resistant plants, then use the approaches of PCR to identify the selected resistant plants. 2 masculine plants were obtained. |
