Expression Of Fibroblast Growth Factor 21(FGF21) In Transgenic Tobacco And Safflower

Fibroblast growth factor-21 (FGF21) is a member of the FGF superfamily, which consists of 23 secreted proteins. The highest levels of FGF21 expression are in the liver. FGF21 activates glucose uptake in adipocytes, reduces plasma glucose and triglyceride concentrations. FGF21 is currently of great therapeutic promise as, unlike the other FGFs, it has an excellent safety profile. FGF21 did not show significant mitogenic potential in cell lines. Furthermore, FGF21 administration does not lead to either hypoglycaemia or oedema, which are two common side effects of current diabetes therapies. It was expected to be used for the treatment of insulin resistance of type 2 diabetes. At present, the commonly used prokaryotic expression system, which expressed protein without post-translational processing, like glycosylation, phosphorylation. In this study, we produced tobacco (Nicotiana benthamiana) and safflower (Carthamus tinctorius L.) plants expressing human recombinant FGF21 (hFGF21) via Agrobacterium-mediated transformation using a potato virus X (PVX)-based vector (pGR107) and oil body expression vector pCAMBIA1390Do (p1390Do) respectively, which was a low-cost, convenient, safe, and exact way.hFGF21 gene was designed using preferred codons, which were optimized for expression in dicotyledonous plants. The plant expression vectors pgR107-smGFP-hFGF21 and p1390DohFGF21 was obtained by cloning hFGF21 gene into the pgR107-smGFP and p1390Do vector, respectively. The pgR107-smGFP-hFGF21, pgR107-smGFP, and pgR107-HishFGF21 plasmids were transferred into Agrobacterium tumefaciens strain GV3101 using a freeze-thaw method. Then, the Agrobacterium strains were infiltrated onto the underside of tobacco leaves. After p1390DohFGF21 were transferred into A. tumefaciens strain EHA105, then hFGF21 gene was introduced into safflower (C. tinctorius L.) plants via Agrobacterium-mediated leaf disc transformation. The transgenic tobacco and safflower plants were determind by PCR, RT-PCR and Western blot. The purified hFGF21 from Nicotiana benthamiana stimulated glucose uptake in 3T3/L1 cells were studied. The following main conclusions:1. The plant binary expression vector pgR107-smGFP-hFGF21 was successfully obtained. The recombinant hFGF21 had accumulated to levels as high as 450μg·g-1 fresh weight in leaves of agroinfected plants. The recombinant hFGF21 was purified from plant tissues by Ni-NTA affinity chromatography, and the purified hFGF21 stimulated glucose uptake in 3T3/L1 cells. This indicated that the recombinant hFGF21 expressed via the PVX viral vector in Nicotiana benthamiana was biologically active.2. The plant binary expression vector p1390DoFGF21 that carries hygromycin selection marker was successfully constructed, then the hFGF21 gene was introduced into cultivated Xinjiang safflower (C. tinctorius L.) plants via Agrobacterium-mediated leaf disc transformation. And twenty-two positive safflowers were obtained from one hundred and one transgenic plants, the transformation efficiency value is 21.7% by PCR amplification. The results showed that hFGF21 gene was transferred to safflower plants3. This study successfully resolved rooting difficult problem in safflower using cleft grafting and top grafting method. The results showed that:the survival rate of cleft grafting method is 50%, but the survival rate of top grafting method is 16.7%, so the cleft grafting method is the best.We firstly expressed hFGF21 protein with biological activity using plant virus based vector, which has great significance for plant bioreactor research.