2.2. Plasmid Construct

PCR primers (5′-TGATCCATGGCCTTGACCTTTGCTTTACTG-3′ as the forward primer and 5′-GTGCTCTAGATCATTCCTTACTTCTTAATC-3′ as the reverse primer) were used to amplify human IFN-α 2a (GenBank: {"type":"entrez-nucleotide","attrs":{"text":"JN848522.1","term_id":"357601474"}}JN848522.1). The primers were created with Nco I and Xba I site extensions, respectively. The primer annealing temperature was 56 °C and the MgCl₂ concentration used in the PCR buffer was 2.5 mM. Because IFN-2a genomic DNA sequence contains no introns [24], DNA extracted from human blood was used as a PCR template. IFN-α2a PCR product was column purified (Qiagen, Darmstadt, Germany), then cut with Xba I and Nco I restriction enzymes before being ligated into the pTRA-PT vector (gi13508478). IFN-α2a was then confirmed by sequencing. The scaffold attachment regions (SAR) of tobacco RB7 gene (gi3522871), 5′ UTR of tobacco leader peptide (TL), and 3′ UTR of CaMV 35S (pA35S) flanked the expression cassette of the IFN-α2a gene coding sequence. The expression of the IFN-α2a gene is influenced by the CaMV 35S promoter. The selection of transgenic lines of white and red Raphanus sativus L. was performed by spraying the foliar parts of 3 weeks old plantlets with BASTA (phosphinothricin 25 µg/mL) 6 times at 3-day intervals based on the presence of the PAT (phosphinothricin acetyltransferase) expression cassette. The structure of the used binary expression vector was previously described [20] and the structure of the IFNα2a expression cassette is illustrated in Figure 1.

IFN-α2a gene expression construct.pTRA-PT, a derivative of pPAM (gi13508478) with a constitutive CaMV p35SS promoter and the 5′ UTR of the Tobacco Leader peptide (TL) was used for IFN-α2a expression in Raphanus sativus L. plants. RB/LB: right and left border sequences of nopaline-Ti-plasmid pTiT37. pAnos: polyadenylationsignal of nopaline synthetase gene derived from Agrobacterium tumefaciens. PAT: phosphinothricin acetyltransferase gene that confers resistance to BASTA (phosphinothricin). Pnos: nopaline synthase promoter of A. tumefaciens. IFN-α2a expression cassette is flanked by the 3′ UTR of CaMV (pA35S) and the scaffold attachment region (SAR) of the tobacco RB7 gene (gi3522871).