Expression Of Basic Fibroblast Growth Factor (bFGF) In Transgenic Flammulina Velutipes And Tomato

Production of exogenous proteins using edible fungi as bioreactors has recently been the focus of considerable research interest, meanwhile using the plant systems to large-scale product the medical value of various proteins, peptides or antibodies and other drugs has been the dream of the people, and the rapid development of transgenic plants enable this dream becoming a reality. Compared to other bioreactor systems, edible fungi and plants allow the cost-effective production of recombinant proteins on an agricultural scale, while eliminating risks of product contamination with endotoxins or human pathogens. Another advantage of the use of plants in recombinant protein production is that vaccine candidates can be expressed in edible plant organs, allowing them to be administered as unprocessed or partially processed materials.Basic fibroblast growth factor (bFGF) is a multifunctional growth factor. It has enormous clinical application prospects in promoting angiogenesis, wound healing and regulating immune function. At present, the expression of bFGF protein was mainly in E.coli. So, to meet the increasing demand of this expensive eytokine, we expressed it in transgenic plants.Fisrt, A bFGF expression vector p139035S-bFGF was constructed and trasnsformed into Flammulina velutipes via Agrobacterium tumefaciens using a hygromycin phosphotransferase gene (Hyg) as a selective marker.Second, The lowest inhibitory concentrations of hygromycin for F. velutipes were found to be 9μg/mL on PDA solid medium and 6μg/mL in liquid medium, respectively. The toxicity of Cefotaxime to F. velutipes and the critical factors influencing transformation effciency including the concentration of bacterium, infection time, the concentrion of acetosyringone(AS), and the time of co-culture were tested. Our result showed that1) Cefotaxime had no negative effcet on F. velutipes growth and the best selective concentrion was 400μg/mL2) The maximum transformation efficiency was achieved under a condition when the bacterial growth reaching an OD600 value of 0.5, infection time was for about 30min, AS concentration was 200μmol/L and co-cultivation time was 72h3) The result of PCR and Southern blot indicated that bFGF was integrated into the F. velutipes genome 4) After five successive transfers on solid PDA medium without selection pressure, the bFGF gene was still detectable in transgenic F. velutipes strains, suggesting the stable integration of the exogenous gene.Third, We select tomato named ZhongShu 6 for the experimental plant material, and established efficient regeneration system. We transformed the target gene and selectable marker gene into the plant cell by Agrobacterium-mediated, then culture it in the culture medium which contain the selection agent, and screening the regeneration plant which contain the target gene. Finally, some molecular assays are applied on these transgenic tomato lines1) DNA PCR and Southern blot results proved that the target gene is successfully integrated into the genome of tomato2) RT-PCR results indicated that the bFGF gene is expressed at the transcriptional level3) SDS-PAGE and Western blot resulted confirm the expression of target protein in tomato...