RESUMO
The use of a nonlethal selection scheme, most often using the aadA gene that confers resistance to spectinomycin and streptomycin, has been considered critical for recovery of plastid transformation events. In this study, the plastid-lethal markers, glyphosate or phosphinothricin herbicides, were used to develop a selection scheme for plastids that circumvents the need for integration of an antibiotic resistance marker. The effect of selective agents on tobacco (Nicotiana tabacum) mesophyll chloroplasts was first examined by transmission electron microscopy. We found that at concentrations typically used for selection of nuclear transformants, herbicides caused rapid disintegration of plastid membranes, whereas antibiotics had no apparent effect. To overcome this apparent herbicide lethality to plastids, a "transformation segregation" scheme was developed that used two independent transformation vectors for a cotransformation approach and two different selective agents in a phased selection scheme. One transformation vector carried an antibiotic resistance (aadA) marker used for early nonlethal selection, and the other transformation vector carried the herbicide (CP4 or bar) resistance marker for use in a subsequent lethal selection phase. Because the two markers were carried on separate plasmids and were targeted to different locations on the plastid genome, we reasoned that segregation of the two markers in some transplastomic lines could occur. We report here a plastid cotransformation frequency of 50% to 64%, with a high frequency (20%) of these giving rise to transformation segregants containing exclusively the initially nonselected herbicide resistance marker. Our studies indicate a high degree of persistence of unselected transforming DNA, providing useful insights into plastid chromosome dynamics.
Assuntos
Cloroplastos/genética , DNA de Cloroplastos/genética , Glicina/análogos & derivados , Herbicidas/farmacologia , Nicotiana/genética , Aminobutiratos/farmacologia , Cloroplastos/efeitos dos fármacos , Cloroplastos/ultraestrutura , DNA de Cloroplastos/metabolismo , Resistência a Medicamentos , Marcadores Genéticos , Germinação/efeitos dos fármacos , Glicina/farmacologia , Microscopia Eletrônica , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Plantas Geneticamente Modificadas , Plasmídeos/genética , Recombinação Genética , Sementes/efeitos dos fármacos , Sementes/crescimento & desenvolvimento , Espectinomicina/farmacologia , Estreptomicina/farmacologia , Nicotiana/efeitos dos fármacos , Nicotiana/ultraestrutura , GlifosatoRESUMO
Prostaglandins (PGs) can be synthetized via two isoforms of cyclooxygenase (COX). COX-1 is constitutively expressed in normal tissues, and its activity represent the normal physiological output of PGs. In inflammatory states, the newly discovered COX-2 is rapidly induced, and its activity accounts for the large amounts of PGs seen in inflammation. The commercially available nonsteroidal anti-inflammatory drugs (NSAIDs) are nonselective inhibitors of both COX isoforms; therefore, they provide anti-inflammatory activity as well as side effects associated with COX-1 inhibition. Selective inhibition of COX-2 expression explains at least in part the potent anti-inflammatory activity of steroids. Anti-inflammatory activity of newly developed COX-2 inhibitors, such as NS-398 or SC-58125, suggest a new approach of inflammatory diseases with more efficacious NSAIDs essentially devoid of side effects such as stomach ulcers.
