Defensin encoding gene amplification by 3′ RACE, cDNA cloning and direct sequencing

A defensin encoding gene was amplified using a 3′ RACE technique. A full-length sequence was carried out using degenerate primers that were designed using nucleotide sequences from a Fabaceae defensin pre-peptide gene. A PCR reaction was achieved in a total volume of 50 µl containing 10 × reaction buffer, 2 mM dNTP, 25 mM MgSO_4, 10 µM DEF1-Forward primer, 10 µM Oligo dT primer and 1 U of KOD-plus-Neo Taq DNA polymerase enzyme (Toyobo, Osaka, Japan). The nucleotide sequence of primers used are shown in Table 2. The reaction was initially conducted by using a pre-denaturation program at 94 °C for 2 min, followed by 45 cycles of 98 °C for 10 sec, 50 °C for 30 sec, 72 °C for 30 sec, with a final extension at 72 °C for 7 min. The amplified product was used as a template for subsequent amplification using a DEF2-Forward primer with the conditions described above. The amplified product from each plant (approximately 250–450 bp) was cleaned and cloned into a pJET 1.2/blunt vector (Thermo Fisher scientific, Waltham, MA) and transformed into E. coli TOP 10 F according to a standard heat shock technique⁵². From each plant, ten positive clones harboring plasmids pre-screened by a colony-PCR were extracted from the overnight culture using NucleoSpin^® Plasmid purification (Macherey-Nagel, Duren, Germany) and were then sequenced using vector specific primers.

The nucleotide sequence of primers used in this study.

^*Bolds and italics are NdeI and SapI restriction sites, respectively.