Amplification of a serine protease inhibitor gene from Rhamnus Frangula L

Abstract   Protease inhibitors are proteins that are found in plants, humans, animals, and most microorganisms and their function is to regulate the proteolytic activity. serine protease inhibitors have received great interest for their multiple applications in biotechnology and biomedicine and are the most prominent inhibitors in plants. Based on the amino acid information of trypsin inhibitor of kunitz trypsin inhibitor 3 Glycine max (soybean), degenerated primers were designed and a full-length cDNA sequence named RfIP1(Rhamnus frangula Protease Inhibitor 1) was amplified from the leaves DNA RNA by using PCR and RT-PCR respectively. Electrophoresis analysis showed a difference in the migration of the amplicons obtained on DNA and cDNA, revealing the existence of an intron of 210 nucleotides. The result of the RT-PCR proves that the sequence ofcDNA contained an open reading frame (ORF) of 570 bp, encoding 190 amino acids residues. In conclusion, we successfully got DNA and synthesized cDNA of serine protease inhibitor the Kunitz-type from plant Rhamnus frangula L, which adds to other protease inhibitors extracted from plants.