Análisis de la expresión diferencial de genes involucrados en las vías metabólicas responsables de la síntesis de antocianinas en frutos de Fragaria chilensis ssp. chiloensis f. chiloensis y Fragaria chiloensis ssp. chilensis f. patagónica

133 p.El objetivo de la tesis doctoral fue realizar un estudio comparativo de la pigmentación de fruto dentro de las dos formas botánicas de Fragaria chiloensis ssp. chiloensis. El estudio fue abordado a nivel transcripcional y químico. Para ello, frutos de ambas formas botánicas, f. chiloensis (frutilla nativa blanca) y f. patagonica (frutilla nativa roja), fueron recolectados en cuatro distintos estadios de desarrollo y maduración (verde pequeño, verde grande, blanco y maduro). A partir de fruto completo, sus órganos constituyentes (receptáculo y aquenio) y tejidos, se analizaron los perfiles transcripcionales de genes estructurales y regulatorios (factores de transcripción) involucrados en las vías de biosíntesis de compuestos fenilpropanoides responsables de la formación de antocianinas a lo largo de los diferentes estadios. Mediante este exhaustivo análisis transcripcional, se seleccionó uno de los genes regulatorios, FcMYB1, debido a su expresión diferencial detectada entre receptáculo de fruto rojo y blanco. El ortólogo de este gen aislado en frutilla comercial demostró ser un supresor de la acumulación de pigmentos antociánicos en el sistema heterólogo de Nicotiana. Para caracterizar su función de manera homóloga en Fragaria chiloensis ssp. chiloensis f. chiloensis, frutos de frutilla blanca en estadio verde grande fueron agroinyectados con una cepa de A. tumefaciens portadora de una construcción de RNAi para FcMYB1 y cosechados en estadio maduro. Este trabajo demostró que la supresión génica del factor de transcripción FcMYB1 propició la aparición de un fenotipo de frutos más pigmentados que los frutos control agroinyectados con vector vacío. Consecuentemente, los genes flavonoides finales relacionados con la biosíntesis de proantocianidinas mostraron niveles muy bajos de transcritos de leucoantocianidina reductasa (LAR) y antocianidina reductasa (ANR) en contraste con los niveles aumentados de transcritos de genes directamente relacionados con la biosíntesis de pigmentos antociánicos, antocianidina sintasa (ANS) y UDP-glicosiltransfersa (UFGT). Esta redirección a nivel transcripcional de la vía flavonoides coincidió con una mayor acumulación de antocianinas en frutos agroinyectados con la construcción RNAi-FcMYB1 detectadas por HPLC-DAD. En conclusión, por medio del estudio comparativo en frutos contrastantes en pigmentación de una misma especie de frutilla, se detectaron diferencias en los niveles de transcritos de genes estructurales y regulatorios. Tras este análisis se seleccionó un factor de transcripción que demostró ser importante en la producción de pigmentos en esta especie. Los genes diferencialmente expresados (estructurales y/o regulatorios) identificados en esta investigación pueden ser empleados como marcadores moleculares para asistir los programas de mejoramiento genético de frutilla./ABSTRACT: The aim of this doctoral thesis was to perform a comparative study of the fruit pigmentation of two botanical forms of Fragaria chiloensis ssp. chiloensis. The study was carried out at the transcriptional and chemical level. For this purpose, fruits of both botanical forms, f. chiloensis (white native strawberry) and f. patagonica (red native strawberry), were collected and sorted in four distinct developmental and ripening stages (small green, large green, white and ripe). From whole fruit, their constituent organs (receptacle and achene) and tissues, the transcriptional profiles of structural and regulatory (transcription factors) genes involved in anthocyanin related biosynthesis pathways were assessed among the different stages. By means of this exhaustive transcriptional analysis, FcMYB1, a regulatoty gene was selected due to its differential expression detected between the receptacle of red and white fruits. Its orthologous gene isolated from the commercial strawberry demonstrated it to be a suppressor of the anthocyanin pigment as shown by accumulation in the heterologous system Nicotiana. For characterizing its function in a homologue manner in Fragaria chiloensis ssp. chiloensis f. chiloensis, fruits of white strawberry at large green stage were agroinjected with an A. tumefaciens strain bearing a RNAi construct for FcMYB1 and collected at ripe stage. According to this work, the gene suppression of this FT FcMYB1 favored a more pigmented phenotype in these fruits than in control fruits agroinjected with empty vector. Consequently, the final flavonoid genes related with proanthocyanidin biosynthesis showed very low levels of leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR) in contrast to the elevated transcript levels of genes closely related to anthocyanic pigment biosynthesis, anthocyanidin synthase (ANS) y UDP-glycosyltransferse (UFGT). This flavonoid pathway redirection at the transcriptional level coincided with a greater accumulation of anthocyanin in RNAi-FcMYB1 agroinjected fruits assessed by HPLC-DAD. In conclusion, through this comparative study carried out in fruits with contrasting pigmentation in the same strawberry species, differences in transcript levels of structural and regulatory genes were detected. After this analysis, a transcription factor that proved to be important in the pigment production in this specie was selected. Differentially expressed (structural and/or regulatory) genes identified in this research could be used as molecular markers for assisting strawberry breeding programs

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Sloan Digital Sky Survey IV: mapping the Milky Way, nearby galaxies, and the distant universe

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