Structural Analysis of Def2 Gene from Trigonella foenum-graecum by Characterization of Its Genomic and cDNA

Many antifungal peptides including defensins change the fungal membrane potential and ultimately lead to fungal cell death. In this study the genomic DNA from Trigonella foenum-graecum (extracted with CTAB method) and specific primers (MDef-F/R) were used for Def2 containing fragment amplification. The amplified DNA fragment with 750 bp length was cloned into pJET1.2 cloning vector, confirmed by PCR (MDef-F/R primers) and restriction pattern (using XbaI enzyme) and sequenced. Also Def2 cDNA was synthesized with 219 bp length and cloned into pJET1.2. The new construct was confirmed by PCR (MDef-F/R primers) and restriction pattern (using XbaI and XhoI enzymes) and sequenced. Comparison of genomic DNA and cDNA sequences showed that Def2 gene contains one intron with 486 bp length and its ORF codes for a polypeptide with a 72 amino acids length. Alignment of the amino acid sequence of Def2 peptide shows 98.8 to 100% homology with other reported plant defensin sequences deposited in GenBank. These confirmed that genomic DNA and cDNA of Def2 gene could be used for improvement of antifungal activity of Brassica napus.