RESUMEN
Expression of single-chain antibody fragments (scFvs) in the plant cytosol is often cumbersome. It was unexpectedly shown that addition at the C-terminus of the ER retention signal KDEL resulted in significantly improved expression levels. In this report the cytosolic location of the scFv-CK was confirmed, excluding possible mistranslocation to other subcellular compartments. It was shown that expression of several other scFvs was also improved in tobacco protoplasts. In addition expression was improved in transgenic potato. Changing from KDEL to KDEI did not affect the enhanced protein expression level. Addition of the KDEL motif is a simple and straightforward tool to stabilize in planta cytosolic expression of many scFvs.
Asunto(s)
Citosol/metabolismo , Regulación de la Expresión Génica de las Plantas , Fragmentos de Inmunoglobulinas/genética , Región Variable de Inmunoglobulina/genética , Nicotiana/genética , Plantas Tóxicas , Solanum tuberosum/genética , Hidrolasas de Éster Carboxílico/inmunología , Línea Celular , Clonación Molecular , Retículo Endoplásmico/metabolismo , Glicosilación , Hibridomas , Región Variable de Inmunoglobulina/inmunología , Datos de Secuencia Molecular , Oligopéptidos/metabolismo , Péptidos/química , Péptidos/metabolismo , Plantas Modificadas Genéticamente , Señales de Clasificación de Proteína/química , Señales de Clasificación de Proteína/metabolismo , Protoplastos/metabolismo , Solanum tuberosum/metabolismo , Nicotiana/metabolismo , Nicotiana/ultraestructura , Transformación GenéticaRESUMEN
Random amplified polymorphic DNA (RAPD) offers a potential basis for the development of a diagnostic assay to differentiate the potato cyst nematode species Globodera rostochiensis and G. pallida. Nine decamer primers have been tested for their ability to amplify species-specific DNA sequences. Primer OPG-05 produced 2 discrete DNA fragments, which were consistently present in 5 G. rostochiensis populations and absent in 5 G. pallida populations. These fragments were detectable in single females as well as in single 2nd-stage juveniles. Their amplification is extremely efficient, and reproducible over a wide range of template concentrations. One-fifth of a single juvenile is sufficient to generate reproducible RAPD markers. The amplification from single juveniles requires no DNA isolation. The use of a crude homogenate does not impair the polymerase chain reaction.