Molecular Cloning and Characterization of Npr1 Ankyrin Domain from Capsicum Annum L.

摘要

The study aims to determine the genomic structure of the NPR1 ankyrin domain isolated from Capsicum annum cv. Cabai Berangkai and predict its secondary and tertiary structure by homology protein modeling. The three weeks old chili leaves were taken as samples for DNA isolation. The NPR1 gene was amplified using specific primers by Nested and Touch-Down PCR for two rounds. The second-round PCR products were cloned into pGEM T-Easy vector and transformed into E. coli DH5α via heat-shock method. The transformant were verified by colony PCR and sequencing. The sequencing data was used for genomic analysis and to determine the 3D structure of the NPR1 ankyrin domain. The sequence analysis of ankyrin domain between CbNPR1 and AtNPR1 resulted in 62.5% identity and 78.1% similarity. The conserved important amino acid of Cys216 and His334 were also observed. In the secondary structure ankyrin repeat containing helix-and β sheet conformation was observed. These conformations were confirmed by tertiary structures using Ankb 24 protein as a template. In conclusion, all of the results suggested CbNPR1 and AtNPR1 are predicted for having similar structural conformation and biological function in the plant defense system.