Análisis de la expresión global de genes asociados a crecimiento muscular en congrio colorado (Genypterus chilensis) bajo respuesta a estrés por manejo
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2014
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es
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Universidad Andrés Bello
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Licencia CC
Licencia CC
Resumen
La diversificación de la acuicultura en Chile considera la incorporación de nuevos cultivos de peces que permitan posicionar a esta industria como uno de los sectores más importantes de la economía nacional. Dentro de los candidatos propuestos se encuentra el congrio colorado (Genypterus chilensis). Esta especie presenta un alto valor comercial en el mercado producto de la excepcional calidad de su carne blanca. Sin embargo, a pesar de las grandes cualidades que presenta, su proyección comercial se ha visto limitada producto de sus bajas tasas de crecimiento en cautiverio. Considerando que el tejido muscular representa alrededor de un 60% de la masa corporal de un pez, nuestro grupo se ha enfocado en estudiar y entender los mecanismos implicados en el crecimiento y desarrollo muscular en peces, así como también el estudio de variables intrínsecas o extrínsecas asociadas a estos procesos. Dentro de estas variables de encuentra el estrés causado por disturbios físicos relacionados a los métodos de cultivo tales como el manejo (handling stress). El estrés se puede definir como una condición en la cual el equilibrio dinámico de un organismo es perturbado como resultado de la acción de estímulos comúnmente definidos como estresores. A pesar de la extensa bibliografía sobre los efectos de agentes estresantes en el sistema neuro-inmune-endocrino de peces, muchos de los mecanismos moleculares por los cuales el estrés afecta el crecimiento muscular en peces se encuentran pobremente estudiados. Esto se debe principalmente a la falta de información genómica disponible de especies piscícolas.
Considerando los antecedentes anteriormente descritos, en este trabajo proponemos como hipótesis que el efecto del estrés por manejo (Handling stress) induce cambios en la expresión global de genes asociados a crecimiento muscular de congrio colorado (G. chilensis) particularmente causando alteraciones en vías asociadas a atrofia muscular. Para verificar esta hipótesis, como primer objetivo se generó un transcriptoma de referencia secuenciado y anotado de G. chilensis mediante el uso de tecnologías de secuenciación masiva. Este comprende un total de 48.480 secuencias del cual un 44% presenta anotación funcional. Como segundo objetivo, se determinó la expresión diferencial de genes en músculo de G. chilensis bajo estrés. Para ello, se secuenciaron transcritos de G. chilensis sometidos a un protocolo de estrés por manejo para su posterior mapeo en el transcriptoma de referencia previamente generado. Un total de 1062 genes variaron su expresión bajo esta condición, dentro de los cuales se encuentran genes asociados a procesos catabólicos, traducción, diferenciación y proliferación celular. Finalmente, como tercer objetivo se validó la expresión diferencial obtenida in silico de ocho genes involucrados en crecimiento y diferenciación muscular por RT- gPCR. Los genes candidatos fueron: Factor de respuesta a daño de DNA (redd-1), Factor de unión a la proteína 4eiF4 (4ebp-1), foxo, atrogina-1 y subunidad 1 reguladora del proteosoma 26S (psdml), todos ellos involucrados en vías de atrofia muscular, junto con smad-2, myoDi y myoD2, los cuales están involucrados en procesos de diferenciación muscular. Todos los niveles de expresión obtenidos por RT-gPCR se correlacionan con los obtenidos por los análisis de RNAseq.
Los resultados obtenidos en este trabajo demuestran que el estrés por manejo induce cambios en la expresión global de genes en músculo de G. chilensis, particularmente, encontrándose sobreexpresados genes claves que participan en vías de atrofia del músculo esquelético. Por último, el transcriptoma de referencia generado nos proporciona una fuente invaluable de información genómica de G. chilensis que puede ser utilizada para monitorear la expresión global de genes bajo diferentes condiciones de cultivo de esta importante especie de interés comercial.
The diversity of Chilean aquaculture consider the incorporation of new farm fish in order to establish this industry as the most important sector of national economy. Among the candidate fish proposed we found the Red cusk-eel (Genypterus chilensis). This specie has an exceptional flesh quality and high commercial value; however, in spite of the great qualities that present, the economic projection of G. chilensis has been limited due to its low growth rates. Considering that the skeletal muscle represent around of 60% of total weight fish, our group has been interested to understand the mechanisms implicated in skeletal muscle growth, as well as intrinsic and extrinsic factors that regulate this process. Among these variables we found the stress caused by physical alteration on fishes related to farm fish practices such as handling denominated handling stress. Stress may be defined as a condition in which the dynamic equilibrium of an organism or homeostasis is disturbed by stimulus action denominated stressors. In spite of large information about stressor effects in the neuro-immune-endocrine system of fish, many of the molecular mechanisms whereby stress affects the skeletal muscle growth are poor understood., mainly due to lack of genomic information available of farms fish. Taking the above into account, in this work we propose as hypothesis that handling stress induces global gene expression change in skeletal muscle during G. chilensis growth. Particularly, causing alterations in signaling pathways related to muscle atrophy. To conduct this hypothesis, as first objective, we generated a reference transcriptome of G. chilensis using next generation RNA sequencing. Red Cusk-eel transcriptome comprising 48.480 sequences with a 44% has a functional annotation. As a second objective we evaluate the differential expression gene in skeletal muscle of G. chilensis in response to stress. For that purpose, we generated sequence transcript or reads of skeletal muscle of G. chilensis under a handling-stress protocol. Reads were mapped onto the reference transcriptome previously generated for the identification of differential expressed genes in silico. Results show a total of 1062 genes that are up or down-regulated under handling stress. Of these total of genes we found participant associated to catabolic process, traduction, differentiation and cellular proliferation. Finally as a third objective, we validate the differential expression obtained in silico of eight candidate genes involved to growth and differentiation of skeletal muscle by RT-gPCR. The candidate genes were: DNA damage-inducible transcript 4 protein (redd-1), Eukaryotic translation initiation factor 4E-binding protein 1 (4ebp-1), foxo, atrogin-1 and 26S proteasome regulatory subunit SI (psmdl), all of them involve in atrophy signaling pathway; in addition of smad2, myoD 1 and myoD 2 which are involve in differential skeletal muscle process. All expression levels obtained by RT-gPCR were correlated with RNAseq analysis. Results obtained in this work reveals that handling stress stimulus induce change on global gene expression in skeletal muscle during G. chilensis growth. Particularly, we identified some key participants associated to skeletal muscle atrophy with differential expression under these conditions. Finally, the G. chilensis reference transcriptome generated in this work provide us invaluable genomic information that could be used to monitoring gene expression associated to different farms conditions of this important economic fish.
The diversity of Chilean aquaculture consider the incorporation of new farm fish in order to establish this industry as the most important sector of national economy. Among the candidate fish proposed we found the Red cusk-eel (Genypterus chilensis). This specie has an exceptional flesh quality and high commercial value; however, in spite of the great qualities that present, the economic projection of G. chilensis has been limited due to its low growth rates. Considering that the skeletal muscle represent around of 60% of total weight fish, our group has been interested to understand the mechanisms implicated in skeletal muscle growth, as well as intrinsic and extrinsic factors that regulate this process. Among these variables we found the stress caused by physical alteration on fishes related to farm fish practices such as handling denominated handling stress. Stress may be defined as a condition in which the dynamic equilibrium of an organism or homeostasis is disturbed by stimulus action denominated stressors. In spite of large information about stressor effects in the neuro-immune-endocrine system of fish, many of the molecular mechanisms whereby stress affects the skeletal muscle growth are poor understood., mainly due to lack of genomic information available of farms fish. Taking the above into account, in this work we propose as hypothesis that handling stress induces global gene expression change in skeletal muscle during G. chilensis growth. Particularly, causing alterations in signaling pathways related to muscle atrophy. To conduct this hypothesis, as first objective, we generated a reference transcriptome of G. chilensis using next generation RNA sequencing. Red Cusk-eel transcriptome comprising 48.480 sequences with a 44% has a functional annotation. As a second objective we evaluate the differential expression gene in skeletal muscle of G. chilensis in response to stress. For that purpose, we generated sequence transcript or reads of skeletal muscle of G. chilensis under a handling-stress protocol. Reads were mapped onto the reference transcriptome previously generated for the identification of differential expressed genes in silico. Results show a total of 1062 genes that are up or down-regulated under handling stress. Of these total of genes we found participant associated to catabolic process, traduction, differentiation and cellular proliferation. Finally as a third objective, we validate the differential expression obtained in silico of eight candidate genes involved to growth and differentiation of skeletal muscle by RT-gPCR. The candidate genes were: DNA damage-inducible transcript 4 protein (redd-1), Eukaryotic translation initiation factor 4E-binding protein 1 (4ebp-1), foxo, atrogin-1 and 26S proteasome regulatory subunit SI (psmdl), all of them involve in atrophy signaling pathway; in addition of smad2, myoD 1 and myoD 2 which are involve in differential skeletal muscle process. All expression levels obtained by RT-gPCR were correlated with RNAseq analysis. Results obtained in this work reveals that handling stress stimulus induce change on global gene expression in skeletal muscle during G. chilensis growth. Particularly, we identified some key participants associated to skeletal muscle atrophy with differential expression under these conditions. Finally, the G. chilensis reference transcriptome generated in this work provide us invaluable genomic information that could be used to monitoring gene expression associated to different farms conditions of this important economic fish.
Notas
Tesis (Bioquímico, Magíster en Bioquímica)
Esta tesis fue financiada por el proyecto FONDAP N° 15110027
Esta tesis fue financiada por el proyecto FONDAP N° 15110027
Palabras clave
Estrés en Peces, Congrios, Desarrollo, Genética, Investigaciones.