An antimicrobial peptide identification from Medicago sativa sazova cultivar and evaluation of its gene expression

In recent years, it is necessary to discovery of new antimicrobial agents because of the increase in antibiotic resistance, the development of herbicide and fungucide resistance, and the harmful effects of these drugs on human health. As new antimicrobial agents, antimicrobial peptides (AMP) stand out with their stable structure and non-toxicity to mammalian cells. It is known that plant seeds, in particular, contain many AMPs. In this thesis study, it was aimed to identify a new termostable AMP from the M. sativa Sazova cultivar and to characterize this AMP. For this purpose, peptides (10 kDa≥) were obtained from Sazova seeds. Disc diffusion, gel overlay assay and microdilution tests were performed for the resulting peptides and it was determined that they contained an antimicrobial peptide. Then, the peptide band in the range that gave the inhibition zone in the gel overlay assay was determined by LC-MS/MS as M. sativa Defensin 2.1, which is a hypothetical peptide with a molecular weight of 5.2048 kDa. Then, to determine the gene and amino acid sequences of the Sazova cultivar Defensin 2.1, the sequences were obtained by cloning into the pGEMT-easy vector and using the Sanger method. It was determined that there are 3 different amino acid differences between the UniProt Defensin 2.1 and Sazova Defensin 2.1 sequences. A new AMP was characterized by predicting the physiochemical properties (Expast ProtParam), secondary (PROCHECK) and tertiary (SwissModel, Phyre2 and PRISPRED) structure of Sazova Defensin 2.1 with in silico tools.