Efecto del primer intrón del gen EVP1 de Eucalyptus globulus sobre la expresión del gen reportero GUS en Arabidopsis thaliana
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Fecha
2016
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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
Resumen
En Chile la industria forestal es la segunda industria más importante en relación al PIB
nacional, representando E. globulus el 21% de las especies plantadas comercialmente. En la
actualidad, la disponibilidad de sitios óptimos para su plantación es baja, es por esto que los
suelos ubicados en el norte de Chile pueden ser una alternativa. Sin embargo, estos suelos se
caracterizan por una alta salinidad y baja disponibilidad de agua.
El sodio es tóxico para la planta si alcanza altas concentraciones intracelulares. Una forma
por la cual la célula resiste altas concentraciones de sodio es a través de su compartimentalización
en la vacuola utilizando una gradiente de protones. Existen dos proteínas claves para la
generación de esta gradiente, la H+-ATPasa vacuolar y la H+-pirofosfatasa vacuolar. En relación a
la H+-pirofosfatasa ha sido reportado que su sobreexpresión aumenta la tolerancia a estrés
osmótico y salino.
Nuestro laboratorio ha aislado el gen de H+-pirofosfatasa vacuolar de E. globulus
(EVP1). La estructura génica presenta una región promotora de alrededor de 2000 pb, además de contener 5 exones y 4 intrones, en el cual el tamaño del primer intrón es 10 veces mayor que el resto de los intrones. Interesantemente, ha sido reportado que intrones de mayor tamaño cercanos a la región 5’, están relacionados con la expresión del gen. Para confirmar este antecedente en E. globulus se generaron construcciones moleculares en las cuales el intrón estuviera en distintos contextos moleculares, todas fusionadas con el gen reportero GUS. Estas construcciones fueron utilizadas para transformar de manera estable plantas de A. thaliana.
En este trabajo se pudo determinar que el primer intrón de EVP1 funciona como un
promotor autónomo, produciendo una expresión tejido específica en polen de A. thaliana.
Además, este intrón no participa en la regulación transcripcional del gen EVP1 frente a una
condición de salinidad, sequía o ABA.
In Chile the forestry industry is the second most important industry according to Chile’s GDP, representing E. globulus 21% of the species planted commercially. Currently, the availability of optimal plantation sites is low, therefore sites located in the northern part of Chile can be an alternative. However, their soils are characterized by high salinity and low water availability. Sodium is toxic for the plant if it reaches high intracellular concentrations. One mechanism by which the cell resists high sodium concentrations is through its compartmentalization in the vacuole using a proton gradient. There are two keys proteins that generate this gradient, the vacuolar H+-ATPase and vacuolar H+-pyrophosphatase. Regarding the latter, it has been reported that its overexpression increases tolerance to osmotic and salt stress. Our laboratory has isolated the H+-pyrophosphatase vacuolar gene (EVP1) from E. globulus. This gene is characterized by having a promoter region of about 2000 bp, and it contains 5 exons and 4 introns, where the first intron is 10 times larger than the rest of the introns. It has been reported that large introns near the 5’ end are related to gene expression. To verify this we built different constructs fusing the intron to the GUS reporter gene. These constructs were used for stable transformation of A. thaliana plants, which were subjected to salt stress, osmotic stress and ABA. In this study we determined that the first intron of EVP1 works as an independent promoter, producing tissue specific expression in A. thaliana pollen. Furthermore, this intron was not involved in the transcriptional regulation of EVP1 in plants subjected to salt stress, osmotic stress or ABA.
In Chile the forestry industry is the second most important industry according to Chile’s GDP, representing E. globulus 21% of the species planted commercially. Currently, the availability of optimal plantation sites is low, therefore sites located in the northern part of Chile can be an alternative. However, their soils are characterized by high salinity and low water availability. Sodium is toxic for the plant if it reaches high intracellular concentrations. One mechanism by which the cell resists high sodium concentrations is through its compartmentalization in the vacuole using a proton gradient. There are two keys proteins that generate this gradient, the vacuolar H+-ATPase and vacuolar H+-pyrophosphatase. Regarding the latter, it has been reported that its overexpression increases tolerance to osmotic and salt stress. Our laboratory has isolated the H+-pyrophosphatase vacuolar gene (EVP1) from E. globulus. This gene is characterized by having a promoter region of about 2000 bp, and it contains 5 exons and 4 introns, where the first intron is 10 times larger than the rest of the introns. It has been reported that large introns near the 5’ end are related to gene expression. To verify this we built different constructs fusing the intron to the GUS reporter gene. These constructs were used for stable transformation of A. thaliana plants, which were subjected to salt stress, osmotic stress and ABA. In this study we determined that the first intron of EVP1 works as an independent promoter, producing tissue specific expression in A. thaliana pollen. Furthermore, this intron was not involved in the transcriptional regulation of EVP1 in plants subjected to salt stress, osmotic stress or ABA.
Notas
Tesis (Magíster en Biotecnología)
Esta tesis se realizó en el Centro de Biotecnología Vegetal de la Facultad de Ciencias Biológicas de la Universidad Andrés Bello y fue financiada por Fundación Ciencia & Vida a través del Proyecto PFB-016.
Esta tesis se realizó en el Centro de Biotecnología Vegetal de la Facultad de Ciencias Biológicas de la Universidad Andrés Bello y fue financiada por Fundación Ciencia & Vida a través del Proyecto PFB-016.
Palabras clave
Suelos Salinos, Eucaliptus Globulus, Arabidopsis Thaliana