Resistencia transgénica para el control del virus motoso del enanismo de la frambuesa (raspberry bushy dwarf virus-rbdv)
Transgenic resistance for the control of raspberry bushy dwarf virus-rbdv
Keywords:
Resistencia transgénica, virus motoso, frambuesa, Agrobacterium tumefaciens, Nicotiana benthamiana, Nicotiana tabacum (es)Transgenic resistance, raspberry bushy, virus, Agrobacterium tumefaciens, Nicotiana benthamiana, Nicotiana tabacum (en)
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RBDV se transmite en asociación con polen y por lo tanto la resistencia genética de algunos cultivares del género Rubus ha sido la única medida de control efectivo. Sin embargo, recientemente apareció la cepa viral R-15 de RBDV, la cual rompió la resistencia natural existente en algunos cultivares comerciales de frambuesa. El principal objetivo de este proyecto fue medir la resistencia transgénica a la infección con RBDV, la cual podría conferirse mediante diferentes secuencias de genes pertenecientes a este virus, adicionalmente, otro objetivo fue entender los mecanismos moleculares de resistencia transgénica que podrían ser generados por estos genes. Para medir la resistencia transgénica a RBDV, se introdujeron siete construcciones conteniendo secuencias génicas de RBDV dentro de Agrobacterium tumefaciens mediante conjugación de la bacteria («triparental mating») y los cultivos resultantes se usaron para transformar fragmentos de hojas de Nicotiana tabacum o fragmentos de tallo de plantas de Nicotiana benthamiana provenientes de invernadero o de cultivo de tejido. Se utilizaron plantas regeneradas a partir de cultivo de tejido en medio que contenía kanamicina para obtener semillas que dieron origen a plantas de la generación F1, las cuales se evaluaron para medir su resistencia a RBDV mediante la inoculación manual de hojas jóvenes y se analizaron para determinar el título de virus mediante ELISA después de 5 y 10 días post-inoculación. Se analizaron más de 20 líneas por cada construcción y los resultados mostraron que cada transgen introducido tuvo algún efecto de protección contra el virus. De esta manera, se obtuvo evidencia de la resistencia inducida por los genes correspondientes a la cápside, la antisecuencia y la polimerasa de RBDV, los cuales dieron diferentes grados de protección. La construcción pJAD-10 (secuencia de la cápside de la cepa de RBDV R-15) generó el más alto nivel de protección entre las construcciones analizadas y algunas líneas de Nicotiana benthamiana transformadas con esta construcción mostraron inhibición o retardo en la aparición de síntomas de la infección con RBDV. Por otra parte, el resultado de la infección de líneas transgénicas transformadas con una secuencia no traducible de la cápside o con la anti-secuencia de ésta, sugiere que la resistencia puede ser generada o mediante la expresión de la proteína o por medio de transcriptos de RNAm para esta proteína. Estas construcciones están ahora disponibles para ser introducidas en frambuesa.
RBDV is transmitted in association with pollen and genetic resistance has been the only effective control measure. However, recently a virus strain (R-15) has appeared which can overcome the viral resistance of most modern raspberry cultivars. A solution to this problem could be the development of transgenic resistance. The main objective of this project was to assess the resistance to RBDV infection that would be conferred by different RBDV gene sequences and to understand the mechanisms of resistance generated by these gene sequences. To assess the RBDV transgenic resistance, seven constructs containing RBDV gene sequences were introduced into Agrobacterium tumefaciens by triparental mating and the resulting cultures were used to transform leaf pieces from plants of glasshouse grown Nicotiana tabacum or in vitro-cultured N. benthamiana. Plants regenerated from kanamycin-resistant callus were allowed to set seed and seedlings of the F1 generation were tested for resistance to RBDV by manually inoculating expanded leaves and assaying for virus titre by ELISA 5 and 10 days after inoculation. More than 20 lines were obtained from transformations with each type of construct and the results showed that transformation with each construct had some effect on virus multiplication. Evidence of resistance induced by coat protein (CP) sense, CP anti-sense and polymerase gene sequences was obtained and several degrees of effectiveness of protection were identified among these constructtransgenes. Construct pJAD-10 (RBDV R-15 CP gene sequence) induced the highest level of protection and some N. benthamiana lines transformed with this construct also showed inhibition or delay in the development of the symptoms of infection by RBDV. Also, the results of challenging transgenic Nicotiana lines transformed with untranslatable CP and CP antisense sequences suggest that resistance can be generated by the expression of RBDV CP and also by mRNA CP transcripts. These constructs are now available to be inserted into raspberry germplasm to provide resistance against R-15 strains of RBDV.
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Copyright (c) 1998 Revista Colombiana de Biotecnología
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