Isolation and Sequencing of Genes Encoding Three Ficin Isoenzymes from Ficus carica Trees of Estahbanat Area of Iran

Background and Aim: The Ficin enzyme, also known as Ficus protease and Debriscine, is a thiol protease from the cysteine family of plant endopeptidase proteases, found in the raw leachate of fig fruit and in the fig branches of the Iranian fig (green form of Ficus carica).The enzyme, which is a single chain polypeptide with an approximate weight of ۲۳-۲۵ kDa, has a cysteine amino acid at its active site. And has three disulfide bonds.The Ficin enzyme, with its known and different isoforms, performs its protease activity at a wider pH and temperature range than other protease enzymes of the same homology. The advantage of this enzyme is its widespread usages in many fields, including industrial, food, pharmaceutical and medical diagnostics. And every year, huge costs are spent on extracting and collecting high-purity enzyme through the latex of fig trees. Therefore, in this study, to take a step towards further genetic research on this useful enzyme. In this research, the genomic content from existing plant samples was extracted, amplified and then the isoforms of that were sequenced Methods: In this study, fresh leaf samples were collected from the green form trees of the Iranian vegetation area and their total-RNA content was extracted according to the Cunninghamia protocol. Then its cDNA was made by Thermo scientific cDNA synthesis kit protocol. So different Ficin enzymes were amplified by degenerated-designed primers. Finally, three samples of the products were sent for sequencing Results: The total RNA content was extracted. The cDNA was produced. Some isoforms were amplified. Three of the the isoforms were sequenced and they were registered in the NCBI data bank Conclusion: In conclusion,RNA extraction,cDNA synthesizes were done successfully,then three isoforms of Iranian Ficin enzyme were amplified due to perform other actions on it to improve its properties for its especial applications.

Keywords:

Protein Isoforms , Latex , Gene Isolation , Sequencing