Expression of a recombinant antimicrobial peptide MsDeF۱.۲ in Nicotiana tabacum

Background and Aim: Antimicrobial peptides (AMPS) are small cationic peptides that show biological activity against a wide range of organisms. They are an innate defense system of organisms. These peptides have low toxicity to animal and plant cells and do not induce resistance due to specific function in pathogen cells. AMPS are a suitable alternative for chemical poisons and conventional antibiotics. MsDeF۱.۲ is an antimicrobial peptide first extracted from Medicago sativa. The aim of this study was to express and produce MsDeF۱.۲ antimicrobial peptide in the final host, tobacco.Methods: Transgenic plants were obtained by co-culture of leaf discs of Nicotiana tobacco L. cv. Xanthi with Agrobacterium tumefaciens carrying the binary vector pBI۱۲۱-MsDeF ۱.۲-HIS. To transform Agrobacterium, freez-thaw method was used to achieve the correct gene transfer to A. tumefaciens. Expression level of MsDeF۱.۲ evaluated by RT-PCR. To ensure the MsDeF۱.۲ peptide production, the ELISA reaction was used and the change in color of the solution from colorless to blue indicated the presence of the peptide and the proper binding of the antibody. The ELISA reader was also used to measure the absorbance of the samples.Results: In this study we used the binary vector pBI۱۲۱ that is one of the most common vectors for Agrobacterium-mediated gene transfer. Presence of CaMV۳۵S promoter in this vector caused over expression of the gene in transgenic plants. The Kozak sequence insert upstream of the gene enabled increased expression of the downstream gene. Codon optimization carried out based on the final host which was tobacco. Presence of ۲۸۳ bp fragments in transformed plant confirmed by PCR with specific primers of MsDeF۱.۲ gene. Five samples of transformed plants were used for RT- PCR. Results showed that the foreign gene has been transcribed in the five samples. Moreover, ELISA analysis of transformed plants from the total protein extraction of leaves confirmed the expression of MsDeF۱.۲ that was specifically recognized by conjugated anti-His antibody.Conclusion: PCR and RT-PCR technique confirmed the gene transfer and expression, and then the production of recombinant peptide by ELISA was confirmed by a histidine antibody. Conducting the experiment at in vivo condition is the next aim for this investigation.

Keywords:

Antimicrobial peptides (AMPS) , Plant defensins , MsDeF۱.۲ , Agrobacterium tumefaciens