Caracterización genética y transcripcional de genes involucrados en la biosíntesis de monoterpenos presentes en el aroma moscatel durante el desarrollo de Vitis vinifera
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2016
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es
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Universidad Andrés Bello
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Resumen
El mercado de la fruta es fundamental para la industria exportadora y la economía de Chile que comercializa alrededor de 40 variedades de uva de mesa, de ellas ninguna posee un carácter organoléptico atractivo y característico como el aroma moscatel, en gran medida debido a la aparente asociación negativa entre el aroma Moscatel y atributos de calidad.
Está demostrado funcionalmente que la acumulación de monoterpenos volátiles característicos del aroma Moscatel, es dependiente del gen que codifica para la 1-desoxi-d-xilulosa-5-fosfato sintasa (DXS), primera enzima de una ruta metabólica que precede a la de estos compuestos aromáticos. Sin embargo, la complejidad e intensidad del aroma depende de la abundancia relativa de cada uno de los monoterpenos que componen el aroma y poco se conoce a nivel genético o funcional.
Durante el desarrollo de esta tesis se trató de dilucidar parcialmente la ruta de los monoterpenos, a nivel genético, transcripcional y de acumulación de metabolitos. Se utilizaron variedades con características organolépticas contrastantes; Crimson Seedless, variedad de aroma neutro, y variedades con distintos niveles de aroma Moscatel (Moscatel de Alejandría, Moscatel Amarilla, Torontel y Pedro Jiménez). En ellas se buscó establecer una asociación entre el genotipo definido en base a VvDXS, la acumulación de transcritos de los genes que codifican para la VvDXS, Geraniol Sintasa (GES) y Linalool Sintasa (LIS), y la acumulación de sus productos metabólicos: geraniol y linalool respectivamente (monoterpenos característicos del aroma Moscatel).
En Moscatel de Alejandría se identificó, en heterocigosis, la mutación de tipo SNP K284N en el gen VvDXS, descrita como principal responsable del desarrollo del aroma de tipo Moscatel. En base a esta mutación y su secuencia colindante se diseñó un marcador de tipo PCR-HRM que permitió analizar las otras variedades utilizadas además de la progenie experimental F1 (primera generación de plantas producto del cruzamiento) (Moscatel de Alejandría x Crimson Seedless). En Pedro Jiménez no se detectó la mutación K284N, sin embargo, su perfil aromático podría deberse a la acumulación de Linalool, cercana al umbral olfativo, que a su vez podría explicarse por la expresión de VvLIS que es mayor a la detectada en las otras variedades estudiadas. El análisis de asociación entre el genotipo de VvDXS y el fenotipo caracterizado a nivel organoléptico, realizado en la progenie experimental, permitió confirmar la asociación descrita entre el genotipo de VvDXS y la detección sensorial del aroma de tipo Moscatel, pero además, señala la existencia de otros determinantes genéticos capaces de contribuir a la generación del aroma de tipo Moscatel, permitiendo la acumulación de monoterpenos en ausencia de la mutación K284N al igual que en la variedad Pedro Jiménez.
The fruit market is essential for the Chilean export industry and economy. Among the forty commercialized table grape varieties none has an attractive and distinctive organoleptic trait such as the Muscat aroma, mainly due to the apparent negative association between Muscat flavor and other quality related traits. It has been functionally characterized that the accumulation of volatile monoterpenes that compose the Muscat aroma, depends on the gene that encodes a 1-deoxy-D-xylulose 5-phosphate synthase (DXS), the first enzyme from the monoterpene metabolic pathway. However, the complexity and intensity of Muscat flavor depends on the relative abundance of each of the monoterpenes and there is no currently knowledge at genetic or functional level. During the development of this thesis we tried to elucidate partially the main components of the monoterpenes pathway, at genetic, transcriptional and metabolite level. We used varieties that contrast at the organoleptic level: Crimson Seedless, a non-aromatic variety, and varieties with different levels of Muscat aroma (Muscat of Alexandrie, Yellow Muscat, Torontel and Pedro Jiménez). We sought to establish an association between genotype (defined upon VvDXS), accumulation of transcripts of the genes encoding VvDXS, Geraniol synthase (GES) and Linalool synthase (LIS), and accumulation of their metabolic products: geraniol and linalool respectively (characteristic monoterpenes of the Muscat aroma). The SNP mutation K284N, described as the main responsible for the development of Muscat Aroma, was identified in heterozygosis, in Muscat of Alexandrie. Based on the mutation and adjacent sequence, we designed a PCR-HRM marker that allowed us to genotype the other varieties used in this work and and an experimental F1 progeny (fist progeny) derived from the cross of Muscat of Alexandrie x Crimson Seedless. Pedro Jiménez resulted negative for the presence of the K284N mutation, suggesting that the aromatic profile of this variety might be explained by the accumulation of linalool at levels close to the olfactory threshold, which in turn might be explained by a higher transcript accumulation of VvLIS compared to the other varieties. The association analysis, between VvDXS genotype and the phenotype characterized at organoleptic level in the experimental progeny allowed us to confirm the association between VvDXS genotype and phenotype characterized at organoleptic level, but also confirms the existence of other genetic determinants that contribute to the generation of Muscat aroma, enabling the accumulation of monoterpenes in absence of the K284N mutation from VvDXS as reported for Pedro Jiménez.
The fruit market is essential for the Chilean export industry and economy. Among the forty commercialized table grape varieties none has an attractive and distinctive organoleptic trait such as the Muscat aroma, mainly due to the apparent negative association between Muscat flavor and other quality related traits. It has been functionally characterized that the accumulation of volatile monoterpenes that compose the Muscat aroma, depends on the gene that encodes a 1-deoxy-D-xylulose 5-phosphate synthase (DXS), the first enzyme from the monoterpene metabolic pathway. However, the complexity and intensity of Muscat flavor depends on the relative abundance of each of the monoterpenes and there is no currently knowledge at genetic or functional level. During the development of this thesis we tried to elucidate partially the main components of the monoterpenes pathway, at genetic, transcriptional and metabolite level. We used varieties that contrast at the organoleptic level: Crimson Seedless, a non-aromatic variety, and varieties with different levels of Muscat aroma (Muscat of Alexandrie, Yellow Muscat, Torontel and Pedro Jiménez). We sought to establish an association between genotype (defined upon VvDXS), accumulation of transcripts of the genes encoding VvDXS, Geraniol synthase (GES) and Linalool synthase (LIS), and accumulation of their metabolic products: geraniol and linalool respectively (characteristic monoterpenes of the Muscat aroma). The SNP mutation K284N, described as the main responsible for the development of Muscat Aroma, was identified in heterozygosis, in Muscat of Alexandrie. Based on the mutation and adjacent sequence, we designed a PCR-HRM marker that allowed us to genotype the other varieties used in this work and and an experimental F1 progeny (fist progeny) derived from the cross of Muscat of Alexandrie x Crimson Seedless. Pedro Jiménez resulted negative for the presence of the K284N mutation, suggesting that the aromatic profile of this variety might be explained by the accumulation of linalool at levels close to the olfactory threshold, which in turn might be explained by a higher transcript accumulation of VvLIS compared to the other varieties. The association analysis, between VvDXS genotype and the phenotype characterized at organoleptic level in the experimental progeny allowed us to confirm the association between VvDXS genotype and phenotype characterized at organoleptic level, but also confirms the existence of other genetic determinants that contribute to the generation of Muscat aroma, enabling the accumulation of monoterpenes in absence of the K284N mutation from VvDXS as reported for Pedro Jiménez.
Notas
Tesis (Magíster en Biotecnología)
financiamiento Fondef y su proyecto G09i1007 (Desarrollo y aplicación de herramientas de genómica e ingeniería genética para potenciar el fitomejoramiento de vides de mesa)
financiamiento Fondef y su proyecto G09i1007 (Desarrollo y aplicación de herramientas de genómica e ingeniería genética para potenciar el fitomejoramiento de vides de mesa)
Palabras clave
Genes Reguladores, Biosíntesis, Uva Moscatel