RNA de interferencia en nicotiana benthamiana como modelo para el desarrollo de plantas resistentes a virus
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Fecha
2011
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Profesor/a Guía
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Idioma
es
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Universidad Andrés Bello
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Licencia CC
Licencia CC
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RESUMEN: El cultivo de las vides es uno de los de mayor importancia económica
para Chile, sin embargo, se ve afectado por numerosos factores, tanto bióticos
como abióticos. Los virus se consideran entre los patógenos de mayor
importancia para este cultivo y hasta el momento se han descrito más de 60
especies de virus que afectan vides. La mayoría de ellos se transmiten a través
de vectores, como nemátodos o áfidos. Se piensa que las metodologías de
propagación a través de estacas serían las responsables del incremento y
amplia diseminación de estos virus en los últimos años. No existen
antecedentes de resistencia natural en vides frente a la infección por virus y la
búsqueda de nuevas variedades resistentes ha sido blanco de muchos
esfuerzos en este campo de investigación. En este trabajo se describe el
desarrollo de resistencia a virus de vides a través de la inducción de
silenciamiento génico en plantas de tabaco.
Consideramos de gran importancia trabajar con los virus más comunes en
Chile, como son: Grapevine leafroll associated virus 2 y 3, Grapevine virus A,
Grapevine fanleaf virus y Grapevine fleck virus, y utilizar secuencias de aislados
chilenos para inducir el silenciamiento génico contra los virus. Se realizó un
análisis de las secuencias disponibles, se utilizaron mayormente secuencias
obtenidas en estudios previos en el laboratorio y se secuenciaron los virus de
los que no se disponían secuencias de aislados locales. En este trabajo se
presenta el caso de Grapevine fleck virus, para el cual se obtuvo la secuencia
del 70% del genoma de un aislado chileno y los fragmentos codificantes para
los dominios proteicos conservados de la replicasa viral para otros 3 aislados,
con los que se analizó la variabilidad de este virus en Chile.
En este trabajo, analizamos la capacidad de diferentes secuencias virales para
gatillar el silenciamiento génico en plantas, y probamos la habilidad de algunas
de ellas para inducir resistencia frente a la infección con el virus. Se eligieron
fragmentos de las secuencias codificantes para proteínas esenciales para el
virus, como proteínas de replicación, movimiento, cubierta y proteínas de shock
térmico, de los diferentes virus.
En este proceso de elección de las secuencias a utilizar para la inducción del
silenciamiento génico, nos llamó particularmente la atención tres proteínas de
Grapevine leafroll associated virus-3, las que no tienen función putativa
asociada y basados principalmente en la similitud de la organización genómica
de GLRaV-3 con virus de otra familia, planteamos la hipótesis de que pudieran
actuar como supresores del silenciamiento génico. Se desarrolló análisis
bioinformáticos para intentar proponer una función putativa para estas tres
proteínas de GLRaV-3, los que arrojaron resultados significativos sólo para la
proteína p19.6 de GLRaV-3, en la cual se identificó un putativo dominio de
unión de RNA doble hebra del tipo arreglos de α-hélices, lo que sin embargo no
nos permitió asociarle una putativa función por ser un dominio presente en...
ABSTRACT: The grapevines are one of the most economically important crops in Chile; however, they are affected by many factors, both biotic and abiotic. Viruses are among the most important pathogens of this crop and thus far more than 60 species of viruses that affect grapevines have been described. Most of them are transmitted in the field by vectors such as nematodes or aphids, but it is thought that the propagation by cuttings would be responsible for the increase and widespread dissemination of these viruses in the last years. There are no records of natural resistance in grapevine against virus infection and the search for new resistant varieties has been the target of many efforts in this field of research. This thesis describes the development of grapevine virus resistance through induction of gene silencing in tobacco plants. We considered of great importance to work with the major viruses present in Chile, including: Grapevine leafroll associated virus 2 and 3, Grapevine virus A, Grapevine fanleaf virus and Grapevine fleck virus; and use of sequences of Chilean isolates to induce gene silencing against them. We did an analysis of the available sequences, most of the ones we used were obtained in previous studies in the laboratory and we sequenced the viruses that lack in local sequences. In this work we present the case of Grapevine fleck virus, for which we obtained the sequence of 70% of the genome of a Chilean isolate and the fragments coding for conserved protein domains of the viral replicase for other three isolates. In this work, we analyzed the ability of different viral sequences to trigger gene silencing in plants, and tested the ability of some of them to induce resistance against infection with the corresponding virus. We selected fragments of the coding sequences for proteins essential for the virus, such as replication, movement, coat proteins and heat shock proteins of the different viruses. In the comparison of the available sequences for the different viruses, and the choosing of the sequences to be used to induce gene silencing, we realized that the genomic organization of Grapevine leafroll associated virus-3, was quite conserved in the family Closteroviridae; most of the viruses of this family encodes for silencing suppressor proteins, and GLRaV-3 has three proteins with no putative function. We performed bioinformatic analyses trying to propose a putative function for these three proteins of GLRaV-3, which yielded significant results only for the p19.6 protein of GLRaV-3. A putative double stranded RNA binding domain was identified, which however did not allow us to associate a putative function because it is a very promiscuous domain that can be found in proteins of various functions. We analyzed the possible activity as a suppressor of gene silencing for the three proteins under study. The results indicate that none of them act as suppressors of gene silencing in our assays, but revealed that they may be able to interact with each other...
ABSTRACT: The grapevines are one of the most economically important crops in Chile; however, they are affected by many factors, both biotic and abiotic. Viruses are among the most important pathogens of this crop and thus far more than 60 species of viruses that affect grapevines have been described. Most of them are transmitted in the field by vectors such as nematodes or aphids, but it is thought that the propagation by cuttings would be responsible for the increase and widespread dissemination of these viruses in the last years. There are no records of natural resistance in grapevine against virus infection and the search for new resistant varieties has been the target of many efforts in this field of research. This thesis describes the development of grapevine virus resistance through induction of gene silencing in tobacco plants. We considered of great importance to work with the major viruses present in Chile, including: Grapevine leafroll associated virus 2 and 3, Grapevine virus A, Grapevine fanleaf virus and Grapevine fleck virus; and use of sequences of Chilean isolates to induce gene silencing against them. We did an analysis of the available sequences, most of the ones we used were obtained in previous studies in the laboratory and we sequenced the viruses that lack in local sequences. In this work we present the case of Grapevine fleck virus, for which we obtained the sequence of 70% of the genome of a Chilean isolate and the fragments coding for conserved protein domains of the viral replicase for other three isolates. In this work, we analyzed the ability of different viral sequences to trigger gene silencing in plants, and tested the ability of some of them to induce resistance against infection with the corresponding virus. We selected fragments of the coding sequences for proteins essential for the virus, such as replication, movement, coat proteins and heat shock proteins of the different viruses. In the comparison of the available sequences for the different viruses, and the choosing of the sequences to be used to induce gene silencing, we realized that the genomic organization of Grapevine leafroll associated virus-3, was quite conserved in the family Closteroviridae; most of the viruses of this family encodes for silencing suppressor proteins, and GLRaV-3 has three proteins with no putative function. We performed bioinformatic analyses trying to propose a putative function for these three proteins of GLRaV-3, which yielded significant results only for the p19.6 protein of GLRaV-3. A putative double stranded RNA binding domain was identified, which however did not allow us to associate a putative function because it is a very promiscuous domain that can be found in proteins of various functions. We analyzed the possible activity as a suppressor of gene silencing for the three proteins under study. The results indicate that none of them act as suppressors of gene silencing in our assays, but revealed that they may be able to interact with each other...
Notas
Tesis (Doctor en Biotecnología)
Palabras clave
Plantas -- Enfermedades a Virus., Plantas -- Enfermedades y Plagas.