Production Of FGF10 In Transgenic Tobacco Plants

Fibroblast growth factor 10(FGF10),which is a growth factor that promotes the value of epidermal cells,is extremely effective in repairing wounds.During the embryonic development stage,the loss of FGF10 and changes in its content can cause many diseases suddenly and therefore it has very important value for scientific research and medical applications.Due to the very limited content of this growth factors in the natural condition and the clinical practice of FGF10 has been restricted to a great extent due to its high price.Therefore,how to develop the new strategies is the basic consideration to solve this problem,and plant bioreactor is just one of better options to meet the demand.In this work,tobacco plant was used as material to be transformed using FGF10 gene,and the prokaryotic expression system was set as a negative control to investigate the feasibility of tobacco as a high-efficiency transformation platform for the large-scale production of the foreign proteins.The N-terminus of FGF10 gene was fused with locating peptide in endoplasmic reticulum of tobacco,a 6x his-tag coding sequence was added to the 5’ end to form a new sequence named EnhFGF10.The main results are as follows.1.Expression of EnhFGF10 using E.coli system.The prokaryotic expression vector pET3a-EnhFGF10 was constructed,and E.coli BL21(DE3)was used as the host strain to express protein induced by IPTG.After optimization,the parameters of expression system were determined,the induction temperature was 37C°,the IPTG concentration was 500 mmol/L,and the induction time was 8 hr.Based on above-mentioned parameters,the expression of EnhFGF10 by E.coli can reached up to the maximum level of approximately 20%of total soluble protein.2.Expression of EnhFGF10 using the plant bioreactor system.The plant expression vector was constructed with artificially modification to form pCAMIA3301-EnhFGF10 which driven by a constitutive strong promoter CaMV 35S.The vector was transferred to tobacco genome by Agrobacterium-mediated transformation.After selection by Basta agent,more than 600 resistant transformants were finally obtained for further screening.There are 582 positive plants among them detected by Bar test strip,and the PCR-positive plants were 570,indicating high efficiency with 98%of transformation.Finally,six transgenic plants were selected via RT-PCR and ELISA to express the target protein for more than 0.1%of the total soluble protein.3.Compared with target EnhFGF10 protein produced by the prokaryotic E.coli expression system,the EnhFGF10 protein produced by tobacco expression system showed no difference in promoting cell proliferation.Both proteins originated from two different systems can promote the proliferation of NIH3T3 cells.These results demonstrated that the EnhFGF10 protein can be expressed in tobacco plant and this tobacco platform is feasible to efficiently produce EnhFGF10.Meanwhile,the transgenic plants obtained in this experiment can be used as germplasms resources with potential application in the market.It is helpfur for the the subsequent development of FGF10-related products.