Salmon Calcitonin Gene Plant Expression Vector And Genetic Transformation

Calcitonin (CT) mainly be used in curing osteoporosis, all kinds of ostalgia , Paget' S disease , hypercalcemia and etc in clinical practice . The calcitonin being used in clinic is the product that separated and purified from salmon's gill lamella or pig's thyroid gland, but the calcitonin produced in this way is too limited and expensive. Although it has been expressed in the prokaryotic cell system, its application is restricted for its deficiency in modification after translation. Therefore, taking the transgenic plant as a bio-reactor to produce calcitonin in large scale has a great meaning in meeting the increasing demand of calcitonin. The aim of this study was to construct the expression vector of salmon calcitonin (sCT) gene, and to transfer sCT gene into lettuce genome and other crops. The main results are as following:1. The sCT gene coding sequence was obtained from its streptomyes vector pMS680 by restriction enzyme BamHI and HindIII , and be cloned into expression vector pART27 to produce a plant expression vector pART27-sCT. The recombinant plasmid be transferred into Agrobacterium tumafaciens C58 by electroporation successfully.2. Particle bombardment was used to transform sCT gene into carrot callus, ELISA analysis of proteins extracted from callus showed the transient expression of sCT gene has achieved. 3. The effects of different hormone concentration on shoot regeneration from cotyledon explants was investigated, the results show that MS medium supplied with 0.1mg/L 6-BA and 0.05mg/L NAA is the suitable shoot regeneration medium; The experiment on sensitivity of cotyledon to kanamycin and carbenicillin shows the suitable kanamycin concentration for selecting transgenic tissue is 75mg/L and the carbenicillin concentration is 300 mg/L.4. sCT gene was transferred into tobacco and lettuce mediated by Ti plasmid of Agrobacterium tumafaciens. Forty tobacco and 118 lettuce which resistance kanamycin was obtained after sifted on shoot regeneration medium and root induce medium supplied with 75mg/LKm; PCR amplification of genomic DNA from kanamycin resistance tobacco and lettuce leaf tissue shows sCT gene was integrated into the genome of transgenic tobacco and lettuce, 11 transgenic tobacco and 29 transgenic lettuce was obtained .