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Estudio De La Interacción Hospedante-Patógeno Entre El Mal De Río Cuarto Virus (MRCV) Y El Delfácido Transmisor Delphacodes Kuscheli

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Estudio De La Interacción Hospedante-Patógeno Entre El Mal De Río Cuarto Virus (MRCV) Y El Delfácido Transmisor Delphacodes Kuscheli Tesis Doctoral Estudio de la interacción hospedante-patógeno entre el Mal de Río Cuarto virus (MRCV) y el delfácido transmisor Delphacodes kuscheli Maroniche, Guillermo Andrés 2011 Este documento forma parte de la colección de tesis doctorales y de maestría de la Biblioteca Central Dr. Luis Federico Leloir, disponible en digital.bl.fcen.uba.ar. Su utilización debe ser acompañada por la cita bibliográfica con reconocimiento de la fuente. This document is part of the doctoral theses collection of the Central Library Dr. Luis Federico Leloir, available in digital.bl.fcen.uba.ar. It should be used accompanied by the corresponding citation acknowledging the source. Cita tipo APA: Maroniche, Guillermo Andrés. (2011). Estudio de la interacción hospedante-patógeno entre el Mal de Río Cuarto virus (MRCV) y el delfácido transmisor Delphacodes kuscheli. Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Cita tipo Chicago: Maroniche, Guillermo Andrés. "Estudio de la interacción hospedante-patógeno entre el Mal de Río Cuarto virus (MRCV) y el delfácido transmisor Delphacodes kuscheli". Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. 2011. Dirección: Biblioteca Central Dr. Luis F. Leloir, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires. Contacto: [email protected] Intendente Güiraldes 2160 - C1428EGA - Tel. (++54 +11) 4789-9293 Universidad de Buenos Aires Facultad de Ciencias Exactas y Naturales Estudio de la interacción hospedante- patógeno entre el Mal de Río Cuarto virus (MRCV) y el delfácido transmisor Delphacodes kuscheli Tesis presentada para optar al título de Doctor de la Universidad de Buenos Aires en el área: CIENCIAS BIOLÓGICAS Guillermo Andrés Maroniche Director: Mariana del Vas Consejero de Estudios: Esteban H. Hopp Lugar de trabajo: Instituto de Biotecnología, Centro de Investigaciones en Ciencias Veterinarias y Agrícolas, Instituto Nacional de Tecnología Agropecuaria (INTA). Buenos Aires, 2011 INDICE DE CONTENIDOS ABREVIATURAS ..........................................................................................................1 RESUMEN...................................................................................................................3 ABSTRACT ..................................................................................................................5 1. INTRODUCCIÓN ......................................................................................................7 1.1. El Mal de Río Cuarto.......................................................................................................7 1.2. El virus del Mal de Río Cuarto y otros reovirus ............................................................ 11 1.2.1. La familia Reoviridae ...........................................................................................................11 1.2.2. El ciclo replicativo de los reovirus .......................................................................................12 1.2.3. Los reovirus vegetales .........................................................................................................14 El género Fijivirus ......................................................................................................................16 El género Oryzavirus .................................................................................................................22 El género Phytoreovirus............................................................................................................22 1.3. La transmisión del virus del Mal de Río Cuarto............................................................ 23 1.3.1. Características de la transmisión.........................................................................................24 1.3.2. Determinantes moleculares de la transmisión de virus por insectos vectores...................24 CAPÍTULO I ............................................................................................................... 27 2. Objetivos ........................................................................................................... 29 3. Métodos ............................................................................................................ 31 3.1. Aislamiento viral y ensayos de transmisión ................................................................. 31 3.2. Extracción de ARN y síntesis de ADNc.......................................................................... 31 3.3. Aislamiento de secuencias de genes candidatos y diseño de oligonucleótidos .......... 32 3.4. PCR cuantitativa en tiempo real y análisis de datos ....................................................32 4. Resultados y discusión....................................................................................... 33 4.1. Adaptación del protocolo de extracción de ARN a un delfácido individual................. 33 4.2. Aislamiento de secuencias de genes de mantenimiento de Delphacodes kuscheli y diseño de oligonucleótidos específicos............................................................................... 34 4.3. Análisis de la expresión de los potenciales genes de referencia ................................. 35 4.3.1. Análisis de la estabilidad de la expresión de los genes candidatos.....................................36 4.3.2. Determinacion del perfil de expresión de los genes candidato estudiados........................38 CAPÍTULO II .............................................................................................................. 41 2. Objetivos ........................................................................................................... 43 3. Métodos ............................................................................................................ 45 3.1. Técnicas clásicas de biología molecular ....................................................................... 45 3.2. Sistema de clonado Gateway ....................................................................................... 45 3.3. Construcción de los vectores utilizados ....................................................................... 46 3.3.1. Vectores de expresión en insectos de las proteínas de MRCV............................................46 3.3.2. Vectores de destino para obtener fusiones fluorescentes..................................................47 3.3.3. Vectores de expresión de marcadores fluorescentes de referencia...................................47 3.3.4. Vectores de expresión de proteínas de MRCV fusionadas a proteínas fluorescentes........48 3.3.5. Vectores de expresión en bacterias ....................................................................................48 3.4. Cultivo y transfección de células Sf9............................................................................ 49 3.5. Localización subcelular por inmunofluorescencia ....................................................... 49 3.6. Localización subcelular en células vivas....................................................................... 49 3.7. Expresión y purificación de proteínas en bacterias .....................................................50 3.8. Inmunización y obtención de un antisuero anti‐P5‐2.................................................. 51 4. Resultados y discusión....................................................................................... 53 4.1. Estudio funcional de las proteínas de MRCV mediante su localización subcelular en células de insecto ................................................................................................................ 53 4.1.1. Análisis de la localización subcelular de las proteínas de MRCV por inmunofluorescencia53 4.1.2. Diseño y construcción de un juego de vectores para estudios de localización subcelular en células vivas de insecto .................................................................................................................57 4.1.3. Análisis de la localización subcelular de las proteínas de MRCV en células vivas ...............62 4.1.4. Análisis de la colocalización subcelular de diferentes proteínas de MRCV en células vivas75 4.2. Estudio del mecanismo de expresión de P5‐2 ............................................................. 78 CAPÍTULO III ............................................................................................................. 85 2. Objetivos ........................................................................................................... 87 3. Métodos ............................................................................................................ 89 3.1. Construcción de vectores............................................................................................. 89 3.2. Expresión de P9‐1 recombinante en bacterias y purificación...................................... 89 3.3. Ensayos de retardo de la movilidad electroforética (EMSA)........................................ 90 3.4. Ensayos de hidrólisis de ATP ........................................................................................ 91 3.5. Localización subcelular y microscopía de fluorescencia .............................................. 92 3.6. Análisis de interacción por Transferencia de Energía de Resonancia Föster (FRET).... 92 4. Resultados y discusión....................................................................................... 93 4.1. Expresión y purificación de P9‐1 en bacterias ............................................................
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