Isolation, identification and study of defensin gene structure (Def1) in Raphanus sativus

Different studies have demonstrated that the plants, by producing antifungal peptides, such as defencins, cause the pores in the fungal plasma membrane which leads to efflux and influx of cellular components. These changes ultimately lead to fungal cell death.The purpose of this research was to isolate, identify, and study of defencin gene structure in Raphanus sativus by cloning and sequencing. The genomic DNA extraction from Raphanus sativus was achived by CTAB method. Amplification of this gene was performed using the specific primers (RDeff /RDefr). The amplified DNA fragment (356 bp) was cloned into pJET1.2 cloning vector and the constructs were confirmed via enzyme digestion and PCR patterns. Also, the cDNA of this gene, which was about 243 bp, was synthesized after extraction of RNA by specific primers. The amplified fragment of the cDNA was confirmed, cloned in the pJET1. 2 vector and sequenced. Comparison of genomic DNA and cDNA sequences showed that Def1gene contains one intron (113bp) and its open reading frame encodes a peptide with a length of 80 amino acids. Alignment of the amino acid sequence of Def1 shows 90 to 98.8% homology with other reported defensin sequences. The identification and cloning of this gene could be used in future for fungal diseases management.