UNIVERSIDAD DE BUENOS AIRES TESIS MAESTRÍA EN...
Solanum tuberosum is the third most important food crop in the world (FAOSTAT, 2009). This crop is moderately sensitive to salinity conditions; in soils with an electrical conductivity (Ec) higher than 5.9 a loss of yield potential of up to 50% is observed. Saline stress involves osmotic and ionic c...
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| Otros Autores: | |
| Formato: | Tesis de maestría acceptedVersion |
| Lenguaje: | Español |
| Publicado: |
Facultad de Farmacia y Bioquímica
2019
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| Materias: | |
| Acceso en línea: | http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=afamaster&cl=CL1&d=HWA_7017 http://repositoriouba.sisbi.uba.ar/gsdl/collect/afamaster/index/assoc/HWA_7017.dir/7017.PDF |
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I28-R145-HWA_7017 |
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dspace |
| institution |
Universidad de Buenos Aires |
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I-28 |
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R-145 |
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Repositorio Digital de la Universidad de Buenos Aires (UBA) |
| language |
Español |
| orig_language_str_mv |
spa |
| topic |
Estrés salino Papa Solanum tuberosum |
| spellingShingle |
Estrés salino Papa Solanum tuberosum Quintana Arrúa, Silverio Andrés UNIVERSIDAD DE BUENOS AIRES TESIS MAESTRÍA EN... |
| topic_facet |
Estrés salino Papa Solanum tuberosum |
| description |
Solanum tuberosum is the third most important food crop in the world (FAOSTAT, 2009). This crop is moderately sensitive to salinity conditions; in soils with an electrical conductivity (Ec) higher than 5.9 a loss of yield potential of up to 50% is observed. Saline stress involves osmotic and ionic components; an imbalance in the solutes is generated that modifies the K+ / Na+ ratio, thus raising the Na+ concentration in the cytosol. Transient increases in calcium have been reported in response to high salt conditions in numerous studies. Calcium dependent protein kinases (CDPKs) are sensor/transducers of the cation and play a central role in the signaling cascades generated in response to ABA and salt stress. These pathways drastically alter the transcriptome by activating or inhibiting the expression of different genes through the action of transcription factors (TFs). The TFs interact with regulatory elements, which are found in the promoter sequences of the genes. There are more than 80 FT families, of which at least five multigene families, AP2 / EREBP, bZIP, MYB / MYC, NAC, and WRKY can play an important role in the salt /drought stress response.
The potato CDPK2 phosphorylates the ABF TF (bZIP family) involved in abiotic stress. In agreement, RT-qPCR assays indicate that StCDPK2 is induced under conditions of salt stress; and in proStCDPK2::GUS plants, promoter activity increases in response to salt. In the laboratory of Dr. Ulloa, transgenic potato plants (cultivar Desiree) that overexpress the isoform StCDPK2, (35S::CDPK2B:6xHis, lines 2B, 2D and 2E) have been generated.
In order to identify TFs involved in the saline stress response, a bioinformatic search was performed using the RNAseq database available at the potato genomics resource Spud DB (http://solanaceae.plantbiology.msu.edu/pgsc). This allowed us to select the ERF5, WRKY6, 25K, R2R3 myb, Small heat stress protein and Salt responsive protein 2 (SRP2) genes, whose expression is modified under saline (24 h NaCl) or osmotic (24 h mannitol) conditions versus the control condition. The promoter sequences of the TFs identified were analyzed with the RSAT (Regulatory sequence analysis tools) application. Among others, motifs related to ABA-, light- and auxin- mediated responses were identified; as well as the different TFs that bind to these motifs.
We decided to validate the RNAseq data in wild (WT) and transgenic 35S::CDPK2 potato plants grown in vitro for 21 days with 2% sucrose (control) or with the addition of 50 and 150 mM NaCl (salt stress). The biometric data and chlorophyll content were analyzed. The root length of the WT plants was significantly lower than in 2E and 2B plants. In addition, line 2E showed higher stem length than WT. Chlorophyll content was determined in leaves of transgenic and WT plants exposed to 400 mM NaCl; the genetically modified lines presented a higher concentration of total chlorophyll compared to the WT. Based on these data, we analyzed the expression of the selected TFs in WT, 2E and 2B lines exposed to control or saline conditions. In order to perform the RT-qPCR assays, specific oligonucleotides were designed to amplify the partial sequences of the TFs, and the elongation factor 1 alpha (EF-1?) or the Gliceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were used as reference genes when indicated. It was possible to standardize the reaction conditions for WRKY, ERF5 and R2R3 myb. For the 21-day test under control conditions, the expression levels of the TFs were lower in the transgenic plants compared to the WT. Under salinity conditions, we observed changes in the expression of WRKY6 in the WT plants and of R2R3 myb in line 2E.
In this work, a first approach was made to study the TFs under salt stress conditions. Our results suggest that their behavior may vary when facing a prolonged period of stress. In addition, bioinformatic, molecular, physiological and biotechnological tools and statistical data analysis were acquired. |
| author2 |
Fantino, Elisa Inés |
| author_facet |
Fantino, Elisa Inés Quintana Arrúa, Silverio Andrés |
| format |
Tesis de maestría Tesis de maestría acceptedVersion |
| author |
Quintana Arrúa, Silverio Andrés |
| author_sort |
Quintana Arrúa, Silverio Andrés |
| title |
UNIVERSIDAD DE BUENOS AIRES TESIS MAESTRÍA EN... |
| title_short |
UNIVERSIDAD DE BUENOS AIRES TESIS MAESTRÍA EN... |
| title_full |
UNIVERSIDAD DE BUENOS AIRES TESIS MAESTRÍA EN... |
| title_fullStr |
UNIVERSIDAD DE BUENOS AIRES TESIS MAESTRÍA EN... |
| title_full_unstemmed |
UNIVERSIDAD DE BUENOS AIRES TESIS MAESTRÍA EN... |
| title_sort |
universidad de buenos aires tesis maestría en... |
| publisher |
Facultad de Farmacia y Bioquímica |
| publishDate |
2019 |
| url |
http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=afamaster&cl=CL1&d=HWA_7017 http://repositoriouba.sisbi.uba.ar/gsdl/collect/afamaster/index/assoc/HWA_7017.dir/7017.PDF |
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AT quintanaarruasilverioandres universidaddebuenosairestesismaestriaen AT quintanaarruasilverioandres estudiodefactoresdetranscripcionrelacionadosalestressalinoenplantasdepapasolanumtuberosum |
| _version_ |
1807326946118336512 |
| spelling |
I28-R145-HWA_70172024-03-07 UNIVERSIDAD DE BUENOS AIRES TESIS MAESTRÍA EN... Solanum tuberosum is the third most important food crop in the world (FAOSTAT, 2009). This crop is moderately sensitive to salinity conditions; in soils with an electrical conductivity (Ec) higher than 5.9 a loss of yield potential of up to 50% is observed. Saline stress involves osmotic and ionic components; an imbalance in the solutes is generated that modifies the K+ / Na+ ratio, thus raising the Na+ concentration in the cytosol. Transient increases in calcium have been reported in response to high salt conditions in numerous studies. Calcium dependent protein kinases (CDPKs) are sensor/transducers of the cation and play a central role in the signaling cascades generated in response to ABA and salt stress. These pathways drastically alter the transcriptome by activating or inhibiting the expression of different genes through the action of transcription factors (TFs). The TFs interact with regulatory elements, which are found in the promoter sequences of the genes. There are more than 80 FT families, of which at least five multigene families, AP2 / EREBP, bZIP, MYB / MYC, NAC, and WRKY can play an important role in the salt /drought stress response. The potato CDPK2 phosphorylates the ABF TF (bZIP family) involved in abiotic stress. In agreement, RT-qPCR assays indicate that StCDPK2 is induced under conditions of salt stress; and in proStCDPK2::GUS plants, promoter activity increases in response to salt. In the laboratory of Dr. Ulloa, transgenic potato plants (cultivar Desiree) that overexpress the isoform StCDPK2, (35S::CDPK2B:6xHis, lines 2B, 2D and 2E) have been generated. In order to identify TFs involved in the saline stress response, a bioinformatic search was performed using the RNAseq database available at the potato genomics resource Spud DB (http://solanaceae.plantbiology.msu.edu/pgsc). This allowed us to select the ERF5, WRKY6, 25K, R2R3 myb, Small heat stress protein and Salt responsive protein 2 (SRP2) genes, whose expression is modified under saline (24 h NaCl) or osmotic (24 h mannitol) conditions versus the control condition. The promoter sequences of the TFs identified were analyzed with the RSAT (Regulatory sequence analysis tools) application. Among others, motifs related to ABA-, light- and auxin- mediated responses were identified; as well as the different TFs that bind to these motifs. We decided to validate the RNAseq data in wild (WT) and transgenic 35S::CDPK2 potato plants grown in vitro for 21 days with 2% sucrose (control) or with the addition of 50 and 150 mM NaCl (salt stress). The biometric data and chlorophyll content were analyzed. The root length of the WT plants was significantly lower than in 2E and 2B plants. In addition, line 2E showed higher stem length than WT. Chlorophyll content was determined in leaves of transgenic and WT plants exposed to 400 mM NaCl; the genetically modified lines presented a higher concentration of total chlorophyll compared to the WT. Based on these data, we analyzed the expression of the selected TFs in WT, 2E and 2B lines exposed to control or saline conditions. In order to perform the RT-qPCR assays, specific oligonucleotides were designed to amplify the partial sequences of the TFs, and the elongation factor 1 alpha (EF-1?) or the Gliceraldehyde-3-phosphate dehydrogenase (GAPDH) genes were used as reference genes when indicated. It was possible to standardize the reaction conditions for WRKY, ERF5 and R2R3 myb. For the 21-day test under control conditions, the expression levels of the TFs were lower in the transgenic plants compared to the WT. Under salinity conditions, we observed changes in the expression of WRKY6 in the WT plants and of R2R3 myb in line 2E. In this work, a first approach was made to study the TFs under salt stress conditions. Our results suggest that their behavior may vary when facing a prolonged period of stress. In addition, bioinformatic, molecular, physiological and biotechnological tools and statistical data analysis were acquired. Fil: Quintana Arrúa, Silverio Andrés. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Buenos Aires, Argeentina Fantino, Elisa Inés Facultad de Farmacia y Bioquímica Ulloa de la Serna, Rita María Quintana Arrúa, Silverio Andrés 2019-10-04 La papa (Solanum tuberosum) es el tercer cultivo alimenticio más importante del mundo (FAOSTAT, 2009). Este cultivo es moderadamente sensible a condiciones de salinidad y en suelos con una conductividad eléctrica (Ec) mayor a 5,9 se observa una pérdida del potencial de rendimiento de hasta un 50%. El estrés salino involucra componentes osmóticos e iónicos, se genera a partir de un desbalance en los solutos que modifica la relación K+/Na+, elevando así la concentración de Na+ en el citosol. Se han reportado aumentos transitorios de calcio en respuesta a condiciones de alta sal en numerosos trabajos (Qingmei 2013, Schmidt 2013). Las quinasas de proteínas dependientes de calcio (CDPKs) son sensoras/transductoras del catión y desempeñan un papel central en las cascadas de señalización generadas en respuesta a acído abscícico(ABA) y al estrés salino (Muñiz García et al. 2012; Grandellis et al. 2016). Estas vías alteran drásticamente el transcriptoma activando o inhibiendo la expresión de distintos genes a través de la acción de factores de transcripción (FTs). Los FTs interaccionan con elementos reguladores, que se encuentran en las secuencias promotoras de los genes. Hay más de 80 familias de FTs, de las cuales al menos cinco familias multigénicas, AP2/EREBP, bZIP, MYB/MYC, NAC y WRKY, pueden desempeñar un rol importante en la respuesta a estrés por sal/sequía. (Xu 2008; Rushton 1998; Tuteja 2007; Singh D 2015, Gahlaut 2016) La CDPK2 de papa fosforila al FT ABF (familia bZIP) involucrado en estrés abiótico (Muñiz García et al. 2012). En concordancia, ensayos de RT-qPCR indican que StCDPK2 se induce en condiciones de estrés salino; y en plantas proStCDPK2::GUS, la actividad del promotor aumenta en respuesta a la sal. En el laboratorio de la Dra. Ulloa se han generado plantas de papa (cultivar Desiree) transgénicas que sobreexpresan la isoforma StCDPK2 (35S::CDPK2:6xHis, líneas 2B, 2D y 2E). Con el objetivo de identificar FTs involucrados en la respuesta a estrés salino, se realizó una búsqueda bioinformática usando la base de datos de RNAseq disponible en el recurso genómico de papa SpudDB (http://solanaceae.plantbiology.msu.edu/pgsc). Esta nos permitió seleccionar los genes ERF5, WRKY6, 25K, R2R3 myb, Small heat stress protein y Salt responsive protein 2 (SRP2), cuya expresión se modifica bajo estrés salino (24 h de NaCl) u osmótico, (24h de manitol) versus la condición control. Se analizaron las secuencias promotoras de los FTs identificados con la aplicación Regulatory sequence analysis tools?RSAT (Chollier et al 2008). Se encontraron motivos relacionados a respuestas por ABA, luz, auxina entre otros donde a su vez hay distintos FT del tipo myb, ERF, bzip que se unen a ellos. Se propuso validar los datos de RNAseq en plantas de papa salvajes (WT) y transgénicas 35S::CDPK2 cultivadas in vitro durante 21 días con 2% de sacarosa (control) o con la adición de 50 y 150 mM NaCl (tratamiento salino). Se analizaron los datos biométricos y el contenido de clorofila. Se pudo observar que la longitud de raíz de las WT resultó ser significativamente inferior con respecto a las líneas 2E y 2B. Además, la línea 2E presentó mayor longitud del vástago respecto de la WT. Se determinó el contenido de clorofila en discos de hojas de plantas transgénicas y WT expuestos a 400 mM de NaCl o agua (control); las líneas modificadas genéticamente presentaron una mayor concentración de clorofila total con respecto al control. En base a los datos obtenidos se decidió analizar la expresión de los FTs seleccionados en las líneas WT, 2E y 2B cultivadas en condiciones control o expuestas a NaCl por 24 h y 21 días. Se diseñaron oligonucleótidos que amplifican las secuencias parciales de los mismos para realizar ensayos de RT-qPCR y se utilizaron como genes de referencia al Factor de elongación 1 alpha (EF-1 ?) o a la Gliceraldehído-3-fosfato deshidrogenasa (GAPDH). Se logró estandarizar las condiciones de reacción para WRKY, ERF5 y R2R3 myb. En el ensayo con plantas WT expuestas por 24 h a sal no se observaron grandes cambios en los niveles de expresión de los FTs. En el ensayo de 21 días en condiciones control, los niveles de expresión de los FTs fueron menores en las plantas transgénicas en comparación con las WT. Además en condiciones de salinidad se observaron cambios en la expresión de WRKY6 en las plantas WT y de R2R3 myb en la línea 2E. En este trabajo se realizó una primera aproximación al estudio de los FTs en condiciones de estrés salino que indica que su comportamiento puede variar frente a un estrés prolongado en el tiempo. Asimismo, se adquirieron herramientas bioinformáticas, moleculares, fisiológicas y biotecnológicas y se realizó un análisis estadístico de los datos. application/pdf Vazquez Rovere, Cecilia Gallego, Susana Mabel Pettinari, Julia Estrés salino Papa Solanum tuberosum spa Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by-nc-nd/2.5/ar/ Estudio de factores de transcripción relacionados al estrés salino en plantas de papa (Solanum tuberosum) info:eu-repo/semantics/masterThesis info:ar-repo/semantics/tesis de maestría info:eu-repo/semantics/acceptedVersion http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=afamaster&cl=CL1&d=HWA_7017 http://repositoriouba.sisbi.uba.ar/gsdl/collect/afamaster/index/assoc/HWA_7017.dir/7017.PDF |