Determinación de la localización sub-celular de proteínas quinasas específicas de polen PSK1, 2, 3 Y 4 de arabidopsis thaliana
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Fecha
2015
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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
Los granos de polen son el gametofito masculino de las plantas, esenciales para su reproducción
y productividad. En Arabidopsis, el grano de polen maduro está compuesto por tres células: una
gran célula vegetativa y dos pequeñas células espermáticas contenidas en el citoplasma de la
célula vegetativa. Durante la fertilización, el tubo polínico transporta direccionalmente a las
células espermáticas hacia el óvulo para producir el evento de doble fertilización. Actualmente, se
conoce poco acerca de las vías de transducción de señales y mecanismos moleculares
involucrados en el proceso de desarrollo y función del polen. Utilizando datos de microarreglos
se han identificado cuatro genes que codifican para proteínas quinasas en polen, denominados
POLLEN SPECIFIC KINASE1-4 (PSK1-4) que se expresan durante las últimas etapas del
desarrollo del polen, germinación y elongación del tubo polínico en Arabidopsis. Como parte del
proceso de caracterización de dichos genes en el desarrollo del polen, es necesario analizar la
localización sub-celular de las proteínas a las cuales codifican. De acuerdo a análisis
bioinformáticos previos basados en secuencias aminoacídicas, PSK1 (At2g41970) y PSK3
(At5g26150) se encontrarían en el citoplasma, mientras que PSK2 (At5g18910) y PSK4
(At5g12000) en el núcleo. Para comprobar experimentalmente estos datos, se generaron fusiones
traduccionales del ORF de cada PSK a GFP bajo el control de tres promotores: El promotor
específico de polen LeLAT52, el promotor endógeno de cada PSK o el promotor constitutivo 35S.
Una vez obtenidas las construcciones, plantas silvestres de Arabidopsis thaliana fueron
transformadas establemente mediante el método de floral dip (LeLAT52 y promotores endógenos)
y hojas de tabaco (promotor 35S) fueron transformadas de manera transitoria mediante
infiltración, para posteriormente analizar la fluorescencia de GFP al microscopio confocal. Los
análisis en hojas de tabaco indican que PSK1 se localiza en en la pared celular, PSK2 en el
núcleo, PSK3 en la membrana plasmática y PSK4 en los plasmodesmas. Los análisis en tubo
polínico sugieren que PSK1 y PSK3 se localizan en el citoplasma, PSK2 en el núcleo de la célula
vegetativa –de acuerdo a lo obtenido en hojas de tabaco– y PSK4 en endosomas. Tomando en
cuenta que plantas mutantes en los genes PSK muestran fenotipos anormales durante el
crecimiento y guía del tubo polínico, estos resultados podrían entregar información importante
para determinar su rol biológico.
Pollen grains are the male gametophyte of plants, essential for their reproduction and productivity. In Arabidopsis, mature pollen grain is composed of three cells: a vegetative cell and two small sperm cells contained in the cytoplasm of the vegetative cell. During fertilization, the pollen tube directionally transports sperm cells to the egg cell, to produce the double fertilization event. Currently, little is known about the signal transduction pathways and molecular mechanisms involved in the process of development and pollen function. Using microarray data have identified four genes encoding protein kinases in pollen, called POLLEN SPECIFIC KINASE1-4 (PSK1-4) that are expressed during the last stages of the pollen development, germination and pollen tube elongation in Arabidopsis. As part of the characterization of these genes in pollen development, it is necessary to analyze the subcellular localization of the proteins for which they encode. According to previous bioinformatics analysis, based on amino acid sequences, PSK1 (At2g41970) and PSK3 (At5g26150) would be found in the cytoplasm, while PSK2 (At5g18910) and PSK4 (At5g12000) in the nucleus. To experimentally verify this data, we generated translational fusions of the PSKs ORF to GFP under control of three different promoters: the pollen specific promoter LeLAT52, the native promoter of each PSK or the constitutive promoter 35S. Once obtained the constructions, wild type Arabidopsis thaliana plants were stably transformed by floral dip method (LeLAT52 and endogenous promoters) and tobacco leaves (35S promoter) were transiently transformed by infiltration, for subsequent analysis of GFP fluorescence using confocal microscopy. Analyzes in tobacco leaves indicate that PSK1 would be located in the cell wall, PSK2 in the nucleus, PSK3 in the plasma membrane and PSK4 in plasmodesmatas. Analyses in the pollen tube suggest that PSK1 and PSK3 are located in the cytoplasm, PSK2 is localized in the nucleus of the vegetative cell –according to what was obtained in tobacco leaves– and PSK4 in the endosomes. Taking into account that mutants in these PSKs display abnormal phenotypes in pollen tube growth and guidance, these results could provide important information to determine their biological role.
Pollen grains are the male gametophyte of plants, essential for their reproduction and productivity. In Arabidopsis, mature pollen grain is composed of three cells: a vegetative cell and two small sperm cells contained in the cytoplasm of the vegetative cell. During fertilization, the pollen tube directionally transports sperm cells to the egg cell, to produce the double fertilization event. Currently, little is known about the signal transduction pathways and molecular mechanisms involved in the process of development and pollen function. Using microarray data have identified four genes encoding protein kinases in pollen, called POLLEN SPECIFIC KINASE1-4 (PSK1-4) that are expressed during the last stages of the pollen development, germination and pollen tube elongation in Arabidopsis. As part of the characterization of these genes in pollen development, it is necessary to analyze the subcellular localization of the proteins for which they encode. According to previous bioinformatics analysis, based on amino acid sequences, PSK1 (At2g41970) and PSK3 (At5g26150) would be found in the cytoplasm, while PSK2 (At5g18910) and PSK4 (At5g12000) in the nucleus. To experimentally verify this data, we generated translational fusions of the PSKs ORF to GFP under control of three different promoters: the pollen specific promoter LeLAT52, the native promoter of each PSK or the constitutive promoter 35S. Once obtained the constructions, wild type Arabidopsis thaliana plants were stably transformed by floral dip method (LeLAT52 and endogenous promoters) and tobacco leaves (35S promoter) were transiently transformed by infiltration, for subsequent analysis of GFP fluorescence using confocal microscopy. Analyzes in tobacco leaves indicate that PSK1 would be located in the cell wall, PSK2 in the nucleus, PSK3 in the plasma membrane and PSK4 in plasmodesmatas. Analyses in the pollen tube suggest that PSK1 and PSK3 are located in the cytoplasm, PSK2 is localized in the nucleus of the vegetative cell –according to what was obtained in tobacco leaves– and PSK4 in the endosomes. Taking into account that mutants in these PSKs display abnormal phenotypes in pollen tube growth and guidance, these results could provide important information to determine their biological role.
Notas
Tesis (Magíster en Biotecnología)
Proyecto FONDECYT Nº 1120766.
Proyecto FONDECYT Nº 1120766.
Palabras clave
Arabidopsis Thaliana, Proteínas Quinasa, Polen, Biotecnología