{"id":115727,"date":"2018-03-11T10:44:26","date_gmt":"2018-03-11T10:44:26","guid":{"rendered":"https:\/\/www.deberes.net\/tesis\/sin-categoria\/aproximacion-proteomica-y-metabolomica-de-la-produccion-de-metabolitos-secundarios-en-la%c2%adneas-celulares-elicitadas-de-solanum-lycopersicum\/"},"modified":"2018-03-11T10:44:26","modified_gmt":"2018-03-11T10:44:26","slug":"aproximacion-proteomica-y-metabolomica-de-la-produccion-de-metabolitos-secundarios-en-la%c2%adneas-celulares-elicitadas-de-solanum-lycopersicum","status":"publish","type":"post","link":"https:\/\/www.deberes.net\/tesis\/bioquimica-vegetal\/aproximacion-proteomica-y-metabolomica-de-la-produccion-de-metabolitos-secundarios-en-la%c2%adneas-celulares-elicitadas-de-solanum-lycopersicum\/","title":{"rendered":"Aproximaci\u00f3n prote\u00f3mica y metabol\u00f3mica de la producci\u00f3n de metabolitos secundarios en l\u00edneas celulares elicitadas de solanum lycopersicum"},"content":{"rendered":"<h2>Tesis doctoral de <strong> Zuleika Coromoto Brice\u00f1o De Lara <\/strong><\/h2>\n<p>Resumen  el cultivar de tomate mt presenta una serie de caracter\u00edsticas fenot\u00edpicas, como un corto ciclo biol\u00f3gico (inferior a 3 meses) y peque\u00f1o tama\u00f1o (menos de 20 cm), que hacen que sea m\u00e1s adecuado para estudios en laboratorio que otros cultivares de tomate. El objetivo general planteado al iniciar este trabajo fue determinar el comportamiento in vitro del cv. Mt, as\u00ed como llevar a cabo un estudio de los perfiles de metabolitos secundarios y de los patrones de expresi\u00f3n proteica en suspensiones celulares sometidas a la elicitaci\u00f3n con mj, en presencia y en ausencia de dos tipos de ?-Cds: metiladas al azar (cdsm) o hidroxipropiladas al azar (cdsh). El fin \u00faltimo es explorar el potencial de las suspensiones celulares derivadas del cv. Mt para utilizar este sistema en la producci\u00f3n de metabolitos con alto valor a\u00f1adido. Para abordar este objetivo se han utilizado diversas t\u00e9cnicas de an\u00e1lisis cromatogr\u00e1fico (hplc-ms y gc-ms), electrofor\u00e9tico (sds-page e ief), citol\u00f3gico (ttc y fda) y espectrofotom\u00e9trico (determinaciones de metabolitos y de diversas actividades enzim\u00e1ticas); tambi\u00e9n se ha llevado a cabo la microsecuenciaci\u00f3n de fragmentos pept\u00eddicos por nanolc esi ms\/ms y se han utilizado las herramientas bioinform\u00e1ticas correspondientes para el an\u00e1lisis de los resultados. Las suspensiones celulares presentaron una acumulaci\u00f3n de compuestos fen\u00f3licos y triterpenos m\u00e1xima durante la fase de crecimiento exponencial. La elicitaci\u00f3n de las suspensiones celulares con mj y cdsm o cdsh, separadamente o en combinaci\u00f3n, dio lugar a un aumento de los niveles de isofucosterol,  -sitosterol y taraxasterol, si bien los mayores cambios se registraron en los tratamientos con cdsm. Sin embargo, los mayores niveles de fenoles solubles totales se obtuvieron en los tratamientos combinados, sobre todo en el tratamiento cdsm+mj. Adem\u00e1s, ambos elicitores (cds+mj) actuaron de forma sin\u00e9rgica en la producci\u00f3n de \u00e1cido cafeico en el medio intracelular, indicando que la naturaleza fisicoqu\u00edmica de las cds influy\u00f3 en su capacidad para estimular la producci\u00f3n de metabolitos y para actuar como agentes secuestrantes. Por otra parte, en los tratamientos de elicitaci\u00f3n se observ\u00f3, en general, una diminuci\u00f3n de las actividades enzim\u00e1ticas peroxidasa, pal, endoquitinasa y  -1,3-glucanasa en presencia de cdsm y un aumento de dichas actividades enzim\u00e1ticas en presencia de cdsh, en los tratamientos con mj y, sobre todo, en los combinados (cds+mj), lo que indica que las cds y el mj son capaces de regular diferencialmente la actividad de estas enzimas. El an\u00e1lisis del proteoma extracelular de las suspensiones celulares mediante separaci\u00f3n monodimensional en geles de poliacrilamida y microsecuenciaci\u00f3n en las suspensiones elicitadas con cdsm+mj permiti\u00f3 la identificaci\u00f3n de diez fragmentos tr\u00edpticos cuyas secuencias de amino\u00e1cidos mostraron homolog\u00eda con var\u00edas prote\u00ednas pr de s. Lycopersicum, quitinasas, una prote\u00edna asociada a la muerte celular bi\u00f3tica y una peroxidasa cati\u00f3nica. Estas prote\u00ednas podr\u00edan formar parte de las respuestas de defensa en suspensiones celulares de tomate mt. Adem\u00e1s de las prote\u00ednas inducidas en el tratamiento de elicitaci\u00f3n, se identificaron una serie de prote\u00ednas constitutivas, como prote\u00ednas pr del tipo pr-1 y pr-5, endoquitinasas, quitinasas, peroxidasas, una prote\u00edna inhibidora de endoglucanasas f\u00fangicas espec\u00edficas de xiloglucano (xegip) y otras enzimas hidrol\u00edticas implicadas en la ruptura de xilanos y arabinoxilanos como la lexyl2 y la ?-L-arabinofuranosidasa, que pueden estar implicadas en la modificaci\u00f3n de la arquitectura de la pared celular durante el crecimiento del cultivo.                   abstract   the tomato cultivar micro-tom displays several phenotypic characteristics such as small size (less than 20 cm) and a reduced life cycle (less than three months), which make it more suitable for laboratory experiments than standard cultivars. The overall objective proposed in this research is to know the in vitro behavior of micro-tom as well as analyzing the response of mt cell cultures to elicitation with methyl jasmonate (mj) and two  -cyclodextrins (randomly methylated  -cyclodextrins, cdsm, and randomly hydroxypropyl  -cyclodextrins, cdsh), alone or in combination, exploring the changes on both secondary metabolite and protein expression patterns that occur in these cell cultures in the presence of these elicitors. To address this objective, a variety of different techniques including: chromatographic (hplc-ms and gc-ms), electrophoretic (sds-page and ief), cytological (ttc and fda) and spectrophotometric (for the determination of metabolites and enzymatic activities) methods were used. Moreover, tryptic fragments of sds-page bands of interest were analyzed by nanolc esi ms\/ms and the lc-esi-ms\/ms spectra submitted to the database-mining tool for protein identification. An accumulation of both phenolic and triterpenoids compounds was seen during the exponential phase of non-elicited cell suspension cultures. Under elicitation conditions, an increase in the levels of isofucosterol,  -sitosterol and taraxasterol was observed, although the highest levels of these compounds were found in cdsm-treated cells. In contrast, the highest levels of phenolic compounds were detected in the presence of both elicitors, especially in cdsm+mj-treated cells. Moreover, both elicitors act synergistically to stimulate the production of caffeic acid in the intercellular medium, suggesting that the physicochemical properties of cds have a great influence in their ability to induce the production of secondary metabolites and in their metabolite-sequestering properties. On the other hand, a decrease in the enzymatic activities of peroxidase, pal, endochitinase and  -1,3-glucanase was observed in cdsm-treated cells whereas an increase of these enzymatic activities occurred in the presence of cdsh, mj, and in the combined treatments, indicating that both elicitors are able to differentially regulate the activity of these enzymes. Analysis of the extracellular proteome showed the presence of amino acid sequences homologous to several pathogenesis-related (pr) proteins, a cationic peroxidase, and a biotic cell death-associated protein, which suggests that mj and cds could play a role in mediating defense-related gene product expression in mt. Moreover, several constitutive proteins were also identified such as pr 1 and 5 proteins, endochitinases, chitinases, peroxidases, an inhibitor protein of xyloglucan-specific fungal endoglucanase (xegip), and other hydrolytic enzymes like lexyl2 and l-arabinofuranosidase, which are cell wall-degrading enzymes involved in the breakdown of xylans and arabinoxylans, suggesting their involvement in the modification of the cell wall architecture during cell culture growth.           resumen  el cultivar de tomate mt presenta una serie de caracter\u00edsticas fenot\u00edpicas, como un corto ciclo biol\u00f3gico (inferior a 3 meses) y peque\u00f1o tama\u00f1o (menos de 20 cm), que hacen que sea m\u00e1s adecuado para estudios en laboratorio que otros cultivares de tomate. El objetivo general planteado al iniciar este trabajo fue determinar el comportamiento in vitro del cv. Mt, as\u00ed como llevar a cabo un estudio de los perfiles de metabolitos secundarios y de los patrones de expresi\u00f3n proteica en suspensiones celulares sometidas a la elicitaci\u00f3n con mj, en presencia y en ausencia de dos tipos de ?-Cds: metiladas al azar (cdsm) o hidroxipropiladas al azar (cdsh). El fin \u00faltimo es explorar el potencial de las suspensiones celulares derivadas del cv. Mt para utilizar este sistema en la producci\u00f3n de metabolitos con alto valor a\u00f1adido. Para abordar este objetivo se han utilizado diversas t\u00e9cnicas de an\u00e1lisis cromatogr\u00e1fico (hplc-ms y gc-ms), electrofor\u00e9tico (sds-page e ief), citol\u00f3gico (ttc y fda) y espectrofotom\u00e9trico (determinaciones de metabolitos y de diversas actividades enzim\u00e1ticas); tambi\u00e9n se ha llevado a cabo la microsecuenciaci\u00f3n de fragmentos pept\u00eddicos por nanolc esi ms\/ms y se han utilizado las herramientas bioinform\u00e1ticas correspondientes para el an\u00e1lisis de los resultados. Las suspensiones celulares presentaron una acumulaci\u00f3n de compuestos fen\u00f3licos y triterpenos m\u00e1xima durante la fase de crecimiento exponencial. La elicitaci\u00f3n de las suspensiones celulares con mj y cdsm o cdsh, separadamente o en combinaci\u00f3n, dio lugar a un aumento de los niveles de isofucosterol,  -sitosterol y taraxasterol, si bien los mayores cambios se registraron en los tratamientos con cdsm. Sin embargo, los mayores niveles de fenoles solubles totales se obtuvieron en los tratamientos combinados, sobre todo en el tratamiento cdsm+mj. Adem\u00e1s, ambos elicitores (cds+mj) actuaron de forma sin\u00e9rgica en la producci\u00f3n de \u00e1cido cafeico en el medio intracelular, indicando que la naturaleza fisicoqu\u00edmica de las cds influy\u00f3 en su capacidad para estimular la producci\u00f3n de metabolitos y para actuar como agentes secuestrantes. Por otra parte, en los tratamientos de elicitaci\u00f3n se observ\u00f3, en general, una diminuci\u00f3n de las actividades enzim\u00e1ticas peroxidasa, pal, endoquitinasa y  -1,3-glucanasa en presencia de cdsm y un aumento de dichas actividades enzim\u00e1ticas en presencia de cdsh, en los tratamientos con mj y, sobre todo, en los combinados (cds+mj), lo que indica que las cds y el mj son capaces de regular diferencialmente la actividad de estas enzimas. El an\u00e1lisis del proteoma extracelular de las suspensiones celulares mediante separaci\u00f3n monodimensional en geles de poliacrilamida y microsecuenciaci\u00f3n en las suspensiones elicitadas con cdsm+mj permiti\u00f3 la identificaci\u00f3n de diez fragmentos tr\u00edpticos cuyas secuencias de amino\u00e1cidos mostraron homolog\u00eda con var\u00edas prote\u00ednas pr de s. Lycopersicum, quitinasas, una prote\u00edna asociada a la muerte celular bi\u00f3tica y una peroxidasa cati\u00f3nica. Estas prote\u00ednas podr\u00edan formar parte de las respuestas de defensa en suspensiones celulares de tomate mt. Adem\u00e1s de las prote\u00ednas inducidas en el tratamiento de elicitaci\u00f3n, se identificaron una serie de prote\u00ednas constitutivas, como prote\u00ednas pr del tipo pr-1 y pr-5, endoquitinasas, quitinasas, peroxidasas, una prote\u00edna inhibidora de endoglucanasas f\u00fangicas espec\u00edficas de xiloglucano (xegip) y otras enzimas hidrol\u00edticas implicadas en la ruptura de xilanos y arabinoxilanos como la lexyl2 y la ?-L-arabinofuranosidasa, que pueden estar implicadas en la modificaci\u00f3n de la arquitectura de la pared celular durante el crecimiento del cultivo.                   abstract   the tomato cultivar micro-tom displays several phenotypic characteristics such as small size (less than 20 cm) and a reduced life cycle (less than three months), which make it more suitable for laboratory experiments than standard cultivars. The overall objective proposed in this research is to know the in vitro behavior of micro-tom as well as analyzing the response of mt cell cultures to elicitation with methyl jasmonate (mj) and two  -cyclodextrins (randomly methylated  -cyclodextrins, cdsm, and randomly hydroxypropyl  -cyclodextrins, cdsh), alone or in combination, exploring the changes on both secondary metabolite and protein expression patterns that occur in these cell cultures in the presence of these elicitors. To address this objective, a variety of different techniques including: chromatographic (hplc-ms and gc-ms), electrophoretic (sds-page and ief), cytological (ttc and fda) and spectrophotometric (for the determination of metabolites and enzymatic activities) methods were used. Moreover, tryptic fragments of sds-page bands of interest were analyzed by nanolc esi ms\/ms and the lc-esi-ms\/ms spectra submitted to the database-mining tool for protein identification. An accumulation of both phenolic and triterpenoids compounds was seen during the exponential phase of non-elicited cell suspension cultures. Under elicitation conditions, an increase in the levels of isofucosterol,  -sitosterol and taraxasterol was observed, although the highest levels of these compounds were found in cdsm-treated cells. In contrast, the highest levels of phenolic compounds were detected in the presence of both elicitors, especially in cdsm+mj-treated cells. Moreover, both elicitors act synergistically to stimulate the production of caffeic acid in the intercellular medium, suggesting that the physicochemical properties of cds have a great influence in their ability to induce the production of secondary metabolites and in their metabolite-sequestering properties. On the other hand, a decrease in the enzymatic activities of peroxidase, pal, endochitinase and  -1,3-glucanase was observed in cdsm-treated cells whereas an increase of these enzymatic activities occurred in the presence of cdsh, mj, and in the combined treatments, indicating that both elicitors are able to differentially regulate the activity of these enzymes. Analysis of the extracellular proteome showed the presence of amino acid sequences homologous to several pathogenesis-related (pr) proteins, a cationic peroxidase, and a biotic cell death-associated protein, which suggests that mj and cds could play a role in mediating defense-related gene product expression in mt. Moreover, several constitutive proteins were also identified such as pr 1 and 5 proteins, endochitinases, chitinases, peroxidases, an inhibitor protein of xyloglucan-specific fungal endoglucanase (xegip), and other hydrolytic enzymes like lexyl2 and l-arabinofuranosidase, which are cell wall-degrading enzymes involved in the breakdown of xylans and arabinoxylans, suggesting their involvement in the modification of the cell wall architecture during cell culture growth.<\/p>\n<p>&nbsp;<\/p>\n<h3>Datos acad\u00e9micos de la tesis doctoral \u00ab<strong>Aproximaci\u00f3n prote\u00f3mica y metabol\u00f3mica de la producci\u00f3n de metabolitos secundarios en l\u00edneas celulares elicitadas de solanum lycopersicum<\/strong>\u00ab<\/h3>\n<ul>\n<li><strong>T\u00edtulo de la tesis:<\/strong>\u00a0 Aproximaci\u00f3n prote\u00f3mica y metabol\u00f3mica de la producci\u00f3n de metabolitos secundarios en l\u00edneas celulares elicitadas de solanum lycopersicum <\/li>\n<li><strong>Autor:<\/strong>\u00a0 Zuleika Coromoto Brice\u00f1o De Lara <\/li>\n<li><strong>Universidad:<\/strong>\u00a0 Murcia<\/li>\n<li><strong>Fecha de lectura de la tesis:<\/strong>\u00a0 30\/05\/2014<\/li>\n<\/ul>\n<p>&nbsp;<\/p>\n<h3>Direcci\u00f3n y tribunal<\/h3>\n<ul>\n<li><strong>Director de la tesis<\/strong>\n<ul>\n<li>Mar\u00eda  Angeles Ferrer Ayala<\/li>\n<\/ul>\n<\/li>\n<li><strong>Tribunal<\/strong>\n<ul>\n<li>Presidente del tribunal: mercedes Jim\u00e9nez ati\u00e9nzar <\/li>\n<li>Mar\u00eda  de los angeles Bernal pita da veiga (vocal)<\/li>\n<li>Jos\u00e9 Mar\u00eda Obon de castro (vocal)<\/li>\n<li>Mar\u00eda  rosario Castellar rodriguez (vocal)<\/li>\n<\/ul>\n<\/li>\n<\/ul>\n<p>&nbsp;<\/p>\n","protected":false},"excerpt":{"rendered":"<p>Tesis doctoral de Zuleika Coromoto Brice\u00f1o De Lara Resumen el cultivar de tomate mt presenta una serie de caracter\u00edsticas fenot\u00edpicas, [&hellip;]<\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"site-sidebar-layout":"default","site-content-layout":"","ast-site-content-layout":"","site-content-style":"default","site-sidebar-style":"default","ast-global-header-display":"","ast-banner-title-visibility":"","ast-main-header-display":"","ast-hfb-above-header-display":"","ast-hfb-below-header-display":"","ast-hfb-mobile-header-display":"","site-post-title":"","ast-breadcrumbs-content":"","ast-featured-img":"","footer-sml-layout":"","theme-transparent-header-meta":"","adv-header-id-meta":"","stick-header-meta":"","header-above-stick-meta":"","header-main-stick-meta":"","header-below-stick-meta":"","astra-migrate-meta-layouts":"default","ast-page-background-enabled":"default","ast-page-background-meta":{"desktop":{"background-color":"var(--ast-global-color-4)","background-image":"","background-repeat":"repeat","background-position":"center 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