A new vector for chloroplast transformation

M.E. SEGRETIN, S. WIRTH, A. MENTABERRY, F.F. BRAVO-ALMONACID

Boca Chica, República Dominicana

Congreso; Vº Encuentro Latinoamericano y del Caribe de Biotecnología Vegetal; 2004

Redbio

Plant genetic engineering through chloroplast transformation presents several advantages relative to other transformation methods, including higher levels of recombinant protein expression, containment of transgene and feasibility of polycistron expression.  Moreover, transformation results from two homologue recombination events, allowing transgene targeting to predetermined sites in the plastid genome without positional effects.  We developed two plastid transformation vectors named pBSW and pBSW5LUTR carrying expression cassettes including the plastid operon rrn promoter driving the expression of both the selector gene aadA and the transgene of interest.  As transcription terminator we used sequence from the rps16 gene. All these elements were flanked by the appropriate recombinogenic sequence to target the integration to the intergenic region between the genes 16S and trnI. Additionally, pBSW5LUTR has both the promoter and the 5LUTR of the psbA gene upstream the cloning site. To validate both constructions, we introduced the uidA gene and assayed  ƒÀ-glucuronidase activity in two biological systems. These vectors were first tested in Escherichia coli resulting in properly expression of uidA gene. The biolistic method was then employed to transform leaves of Nicotiana tabacum cv. Petit Havana.  After 3-4 rounds of regeneration in RMOP media with 500 mg/L of spectinomicin to reach homoplasty, we evaluated uidA integration in the chloroplast genome (PCR and Southern blot) and b-glucuronidase activity. Both vectors were properly transcribed and translated, being the pBSW5LUTR more efficient than pBSW probably due to higher stabilization of the RNA and/or improved translation of the foreign protein. Thus, we developed two vectors for efficient chloroplast transformation of Nicotiana tabacum, validating insertion into the intergenic region between genes 16S and trnI as a new viable site for integration of transgenes in the plastid genome. pBSW5LUTR is now being used to express different genes and protocols to transform plastids of other plant species with agronomical impact are being assayed.