PLASTID TRANSFORMATION OF A COMMERCIAL POTATO CULTIVAR ALLOWS HIGH LEVELS OF HETEROLOGOUS PROTEIN EXPRESSION

MARÍA EUGENIA SEGRETIN, EZEQUIEL M. LENTZ, SONIA WIRTH, FERNANDO BRAVO ALMONACID

Rosario, Pcia. de Buenos Aires, Argentina

Congreso; XIII Reunión Latinoamericana y XXVII Reunión Argentina de Fisiología Vegetal; 2008

Sociedad Argentina de Fisiología Vegetal (SAFV)

Expression of heterologous proteins in plants has many applications, including molecular pharming and biotic/abiotic stress tolerance. In both cases, high levels of expression are desired. To achieve this goal plastid transformation is an attractive option, but it is well established only for few plants, particularly for tobacco. In previous work we developed a plastid transformation vector named pBSWUTR (Wirth et al, J Biotechnoly 2006) with recombinogenic sequences from tobacco that targets transgene integration between 16S and trnI genes. The selector gene used is aadA wich confers resistance to spectinomycin. Sequence analysis revealed that recombinogenic sequences from tobacco and potato are highly homologous. Transient expression experiments were carried out to set the best parameters for bombardment of two commercial cultivars of S. tuberosum: Desireé and Spunta (Segretin et al., RAFV2006). Bombardment experiments with pBSWUTR carrying the uidA gene (for GUS expression) were conducted in Desireé using a regeneration protocol previously described (Nguyen et al., Plant Science 2005) with minor modifications. We obtained 6 plants (labeled as UTRGus) and many resistant calli that were subjected to histochemical assays for GUS activity. Fifty percent of the plants and calli gave positive results, indicating their transplastomic status, further confirmed by PCR. The plants were transferred to the greenhouse and molecular analyses were carried out. As a control we included tobacco UTRGUS plants previously obtained and characterized in our laboratory. For our surprise Southern blot analysis demonstrated that plants were homoplastic, without the need for several regeneration rounds like in tobacco. Northern blot experiments done on leaf RNA samples confirmed that psbA promoter gave the highest transcript levels. SDS-PAGE stained with Coomassie Brilliant Blue showed levels of GUS expression comparable to rbcL protein (around 20% of TSP), demostrating that pBSWUTR allows high levels of protein expression both in tobacco and potato leaves.