Análisis transcriptómico de hígado de Cyprinus carpio en el proceso de aclimatización estacional
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2017
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es
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Universidad Andrés Bello
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Licencia CC
Resumen
Las respuestas de los organismos a las fluctuaciones ambientales son complejas e involucran múltiples factores, lo cual ha complejizado comprender en su totalidad este fenómeno. Es sabido que esta respuesta no es al azar y que se sustenta en una delicada reprogramación a nivel transcripcional, modelando así una respuesta individual adecuada para cada individuo. Por esta razón, y para visualizar de manera global los cambios transcripcionales concomitantes al proceso de aclimatización estacional del pez Cyprinus carpio (carpa común), en este trabajo hemos evaluado la reprogramación génica de la carpa a los cambios ambientales mediante análisis de RNA-Seq.
En este trabajo, hemos obtenido información transcriptómica de hígado de carpas aclimatizadas usando secuenciación masiva Illumina MiSeq. Se han obtenido 38.012.838 y 34.620.343 lecturas de verano e invierno, respectivamente. Luego del control de calidad las lecturas fueron ensambladas de novo usando el software Trinity, obteniendo un transcriptoma de 239.155 contigs con un N50=1516 pb. Luego de usar la herramienta Blastx con la base de datos de Danio rerio (Uniport) se obtuvieron 79.629 genes los cuales fueron anotados funcionalmente mediante el consorcio de GO (Gene Ontology) según Proceso Biológico (BP), Función Molecular (MF) y Componente Celular (CC). Además, hemos comprobado el análisis bioinformático mediante el uso de RT-qPCR a genes relevantes expresados diferencialmente como APOA1, Prostaglandina D2, RPL4, Vitelogenina y WAP65 obteniendo correlación entre los resultados de RNA-Seq y expresión relativa por PCR cuantitativo.
A partir de la base de datos generada, se revelaron diferentes mecanismos moleculares involucrados en el proceso de aclimatización de la carpa común. Nuestros análisis demostraron que vías metabólicas tales como glucólisis/gluconeogénesis, metabolismo de lípidos, sistema inmune, hipoxia, entre otras, fueron diferencialmente reguladas durante el proceso de aclimatización estacional, poniendo en evidencia la complejidad de los mecanismos de respuesta del organismo de la carpa para enfrentar los cambios medioambientales a los que se encuentra expuesto durante un ciclo estacional.
The response of organisms to environmental fluctuations is complex and involves multiple factors that are mostly unknown, which has become more complex to fully understand this phenomenon. It is known that this response is not random and that is based on a delicate reprogramming at the transcriptional level, and modeling appropriate individual response to each individual. For this reason, and to display globally transcriptional changes accompanying the process of seasonal acclimatization of fish Cyprinus carpio (common carp), in this work we have evaluated the genetic reprogramming of carp to environmental changes by analyzing RNA-Seq. In this work, we have obtained transcriptomic information from liver of acclimatized carp using massive Illumina MiSeq sequencing. For summer and winter, 38,012,838 and 34,620,343 RNA reads where generated respectively. After the quality control step of reads, these were assembled de novo using Trinity software, obtaining a transcriptome of 239,155 contigs with an N50 = 1516 bp. After using the Blastx tool against the Danio rerio database (Uniport), 79,629 genes were obtained, which were functionally annotated using the Gene Ontology (GO) consortium according to Biological Process (BP), Molecular Function (MF) and Cell Component (DC). In addition, we have tested the bioinformatic analysis by using RT-qPCR to relevant genes differentially expressed as APOA1, Prostaglandin D2, RPL4, Vitellogenina and WAP65 obtaining a correlation between the results of RNA-Seq and relative expression by quantitative PCR. From the generated database, different molecular mechanisms involved were revealed in the process of acclimatization of common carp. Our analysis showed that metabolic pathways such as glycolysis / gluconeogenesis, lipid metabolism, immune system, hypoxia, among others, were differentially regulated during the process of seasonal acclimatization highlighting the complexity of the response mechanisms of the carp organism to face the environmental changes that are exposed during a seasonal cycle.
The response of organisms to environmental fluctuations is complex and involves multiple factors that are mostly unknown, which has become more complex to fully understand this phenomenon. It is known that this response is not random and that is based on a delicate reprogramming at the transcriptional level, and modeling appropriate individual response to each individual. For this reason, and to display globally transcriptional changes accompanying the process of seasonal acclimatization of fish Cyprinus carpio (common carp), in this work we have evaluated the genetic reprogramming of carp to environmental changes by analyzing RNA-Seq. In this work, we have obtained transcriptomic information from liver of acclimatized carp using massive Illumina MiSeq sequencing. For summer and winter, 38,012,838 and 34,620,343 RNA reads where generated respectively. After the quality control step of reads, these were assembled de novo using Trinity software, obtaining a transcriptome of 239,155 contigs with an N50 = 1516 bp. After using the Blastx tool against the Danio rerio database (Uniport), 79,629 genes were obtained, which were functionally annotated using the Gene Ontology (GO) consortium according to Biological Process (BP), Molecular Function (MF) and Cell Component (DC). In addition, we have tested the bioinformatic analysis by using RT-qPCR to relevant genes differentially expressed as APOA1, Prostaglandin D2, RPL4, Vitellogenina and WAP65 obtaining a correlation between the results of RNA-Seq and relative expression by quantitative PCR. From the generated database, different molecular mechanisms involved were revealed in the process of acclimatization of common carp. Our analysis showed that metabolic pathways such as glycolysis / gluconeogenesis, lipid metabolism, immune system, hypoxia, among others, were differentially regulated during the process of seasonal acclimatization highlighting the complexity of the response mechanisms of the carp organism to face the environmental changes that are exposed during a seasonal cycle.
Notas
Tesis (Magíster en Biotecnología)
Palabras clave
Carpa, Adaptación, Aspectos Genéticos