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Caracterización de elementos reguladores en cis que responden a estrés iónico y osmótico presentes en el promotor del gen EVP1 de Eucalyptus globulus
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Fecha
2013
Autores
Profesor/a Guía
Facultad/escuela
Idioma
es
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Universidad Andrés Bello
Nombre de Curso
Licencia CC
Licencia CC
Resumen
La industria forestal es un pilar fundamental de la economía chilena, siendo
Eucalyptus globulus la segunda especie más plantada y utilizada en la industria de la
pulpa y papel. El rápido crecimiento de la industria ha llevado a una reducción
significativa de los terrenos aptos para el cultivo, haciendo necesario considerar el uso
de suelos marginales ubicados en el norte de Chile. Sin embargo, estos suelos
presentan sequía y contienen alta salinidad, factores que afectan negativamente la
productividad de los cultivos. Indudablemente, el desarrollo de plantas tolerantes a la
sequía y salinidad permitirían a la industria expandir sus plantaciones, jugando un rol
importante en la prevención de la degradación de los suelos.
En nuestro laboratorio se ha caracterizado funcionalmente una pirofosfatasa
vacuolar de Eucalyptus globulus (EgEVP1) la cual transporta H+ al lumen vacuolar, con
el fin de mantener el gradiente de protones necesario para el intercambio de Na+ a
través del antiporter vacuolar Na+/H+. Sin embargo, no existe información disponible
acerca de su regulación, por lo que el objetivo de este estudio fue caracterizar su región
promotora. Análisis bioinformáticos revelaron la presencia de cuatro elementos
reguladores en cis relacionados a estrés iónico y osmótico: MYCRSRS, MYBRS, ORE y
ABRE. Para estudiarlos, generamos 16 líneas transgénicas de Arabidopsis thaliana
conteniendo el promotor completo y tres deleciones en su extremo 5', fusionadas al gen
reportero GUSA para determinar la funcionalidad de los elementos reguladores en cis
en plantas sometidas a estrés iónico y osmótico.
El ensayo cualitativo de la actividad de GUS indicó que no existen diferencias
entre los tratamientos y el grupo control en todas las construcciones. Además pudimos
identificar una región de 1. 735 pb que representa el promotor mínimo necesario para
activar la transcripción del gen. Por el contrario, el ensayo cuantitativo determinó que el
promotor completo (2.017 pb) es capaz de activar la transcripción del gen reportero de
forma estadísticamente significativa en condiciones normales y estrés osmótico,
respecto a las otras construcciones y plantas silvestres. Este estudio sugiere que la
región de 282 pb del extremo 5' del promotor sería la responsable de la activación del
gen EVP1 ante condiciones normales y estrés osmótico.
The forestry industry is very important to the Chilean economy, being Eucalyptus globulus the second most important tree species used for the pulp and paper industry. The rapid growth of this industry has led to a significant reduction of land suitable for commercial plantations, making it necessary to consider the use of marginal sites located on the northem part of Chile. However, these soils contain high salt concentrations which adversely affect crop productivity. Undoubtedly, the development of salt tolerant plants would allow the industry to expand its plantation area while playing an important role in preventing further land degradation. In our lab we had previously isolated a vacuolar pyrophosphatase from Eucalyptus globulus (EgEVP1) that transports H+ into the vacuolar lumen in arder to maintain the proton gradient necessary to exchange for Na+ through a Na+ /H+ vacuo lar antiporter. However, no information is available about its regulation; therefore we became interested in characterizing its prometer region. Bioinformatics analyses revealed the presence of four cis-acting elements related to ionic and osmotic stress: MYCRSR, MYBR, ORE, and ABRE. To study them, we have generated 16 Arabidopsis thaliana transgenic lines using the complete prometer and three 5' end deletions of the prometer fused to the reporter gene GUSA to establish the functionality of the cis-acting elements in plants subjected to ionic and osmotic stress. Test results of qualitative assay of GUS activity indicate no differences between treatments and the control group in all constructions. Furthermore we identified a region of 1. 735 bp that represent the minimal prometer to activate gene transcription. By contrast, the quantitative assay of GUS activity determined that the complete prometer (2.017 bp) is capable of activating transcription of the reporter gene in a statistically significant way in normal and osmotic stress conditions, compared to the other constrúcts and wild plants. This study suggests that the regían of 282 bp of the 5 'end of the prometer would be responsible for gene activation of EVP1 to normal and osmotic stress.
The forestry industry is very important to the Chilean economy, being Eucalyptus globulus the second most important tree species used for the pulp and paper industry. The rapid growth of this industry has led to a significant reduction of land suitable for commercial plantations, making it necessary to consider the use of marginal sites located on the northem part of Chile. However, these soils contain high salt concentrations which adversely affect crop productivity. Undoubtedly, the development of salt tolerant plants would allow the industry to expand its plantation area while playing an important role in preventing further land degradation. In our lab we had previously isolated a vacuolar pyrophosphatase from Eucalyptus globulus (EgEVP1) that transports H+ into the vacuolar lumen in arder to maintain the proton gradient necessary to exchange for Na+ through a Na+ /H+ vacuo lar antiporter. However, no information is available about its regulation; therefore we became interested in characterizing its prometer region. Bioinformatics analyses revealed the presence of four cis-acting elements related to ionic and osmotic stress: MYCRSR, MYBR, ORE, and ABRE. To study them, we have generated 16 Arabidopsis thaliana transgenic lines using the complete prometer and three 5' end deletions of the prometer fused to the reporter gene GUSA to establish the functionality of the cis-acting elements in plants subjected to ionic and osmotic stress. Test results of qualitative assay of GUS activity indicate no differences between treatments and the control group in all constructions. Furthermore we identified a region of 1. 735 bp that represent the minimal prometer to activate gene transcription. By contrast, the quantitative assay of GUS activity determined that the complete prometer (2.017 bp) is capable of activating transcription of the reporter gene in a statistically significant way in normal and osmotic stress conditions, compared to the other constrúcts and wild plants. This study suggests that the regían of 282 bp of the 5 'end of the prometer would be responsible for gene activation of EVP1 to normal and osmotic stress.
Notas
Tesis (Ingeniería en Biotecnología)
Palabras clave
Eucaliptus Globulus, Genética Forestal, Industria forestal, Uso de suelos marginales, Sequía y salinidad, Chile (Zona Norte)