Development a cloning strategy based on Gateway Technology.

BERTELLI, A; SETTEN, L; NELSON, G; MARCONI, PL; ALVAREZ, MA

Guadalajara

Congreso; REDBIO 2010, VII Encuentro Latinoamericano y del Caribe.; 2010

In order to obtain an efficient DNA fragment single step transference to the Gateway destination vector (pK7WG2D) we have developed a method with direct recombination from PCR products. This strategy was performed because both entry and destination vectors carried the same resistance (Kanamicin). We therefore cloned the gene of interest into the pCR8(GW)TOPO vector (Invitrogen). The process was evaluated using different enzymes: PMT (putrescine N-methyltransferase from Nicotiana tabacum L.) and H6H (Hyoscyamine 6-hydroxylase from Brugmansia x candida Pers.) encoding regions. Consequently, we performed a PCR using M13 forward and reverse primers. This strategy allows the amplification of each cDNA fragment encoding the sequence described above and including attl left and right recombination sequences. Finally, the recombination process was carried out between the extended PCR fragment (containing the attl sites) and the binary plasmid pK7WG2D (Invitrogen). LR Clonase II enzyme MIX was used to catalyze the reaction which was performed according to the manufacturer’s protocol (Invitrogen). Each binary vector obtained was cloned into Agrobacterium tumefaciens EHA101 competent cells by electroporation. Transient expression was performed in tobacco plants for initial studies and provides evidence of the constructs obtained. Each plasmid was isolated and the construction was confirmed by restriction endonuclease map, PCR analysis and sequencing. The sequence was checked using the DNA ABI 373A automated sequencer, according to the Sanger method. All plasmid manipulations were carried out according to standard procedures.