Genes diferencialmente expressos e splicing alternativos relacionados com características de carcaça e carne de bovinos da raça Nelore
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2019-01-25
Autores
Silva, Danielly Beraldo dos Santos [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
O sequenciamento do RNA (RNA-Seq) é uma das abordagens utilizadas para identificar transcritos correspondentes a genes candidatos que provocam variações em vias metabólicas, resultando em diferentes fenótipos. Uma vez que, área de olho de lombo (AOL) e o conteúdo de gordura intramuscular (GI) são características poligênicas, muitos genes e mecanismos que induzem as diferenças no crescimento e desenvolvimento muscular, bem como nos índices de conteúdo de GI da raça Nelore, ainda são desconhecidos. Portanto, o objetivo foi estudar o transcriptoma do músculo de bovinos da raça Nelore, com o propósito de identificar genes e eventos de splicing alternativos diferencialmente expressos (DEGs e DAS) associados à característica de carcaça (AOL) e carne (conteúdo de GI), por meio do RNA-Seq. Para tanto, um total de 80 animais foram sequenciados e fenotipados para AOL e conteúdo de GI (mensurados pelo método químico - que avalia lipídios totais). Os resultados foram apresentados nos capítulos 2, 3 e 4. No capítulo 2, foram selecionados para análise de expressão diferencial, 15 animais com maiores e 15 animais com menores AOL. Após as análises, 288 genes foram diferencialmente expressos (q-value ≤0,05), dos quais 182 foram superexpressos e 106 foram reprimidos no grupo de animais com maiores AOL em comparação com o grupo com menores AOL. Genes pertencentes a famílias da actina, miosina, colágeno, integrina, transportador de soluto, ubiquitina e kelch-like foram diferencialmente expressos. A análise de enriquecimento funcional mostrou que muitos dos Gene Ontology terms (GO) estavam associados com o desenvolvimento, crescimento e degradação muscular. Por meio da análise de rede de co-expressão, foram previstos três genes hub (PPP3R1, FAM129B e UBE2G1), os quais estavam envolvidos no crescimento e proteólise muscular. No capítulo 3, foram selecionados para análise de expressão diferencial, 15 animais com maiores e 15 animais com menores conteúdos de GI. Após as análises, 65 DEGs foram identificados, incluindo 21 genes superexpressos e 44 reprimidos em animais com maiores conteúdos de GI em comparação com animais com menores conteúdos de GI. A análise de enriquecimento funcional mostrou que os GO terms foram associados ao metabolismo lipídico e muscular, assim como ao sistema imune. A análise da rede de co-expressão mostrou que os três genes hub preditos (PDE4D, KLHL30 e IL1RAP) podem desempenhar um papel na biologia lipídica celular e/ou sistêmica em bovinos da raça Nelore. No capítulo 4, foram identificados eventos de splicing alternativos diferencialmente expressos (DAS), e seus genes associados, nos mesmos grupos de animais selecionados no capítulo 2 (fenotipicamente divergentes para AOL) e no capítulo 3 (animais divergentes para conteúdo de GI). Os eventos DAS foram identificados a partir da abordagem exon-centric. Os resultados mostraram 166 e 269 eventos DAS (p-value ≤ 0,05), respectivamente, para AOL e conteúdo de GI. O salto de éxon e sítios alternativos 3’ foram os eventos mais frequentemente encontrados para ambas características. Os DAS encontrados foram transcritos para 125 e 219 genes, respectivamente para AOL e para GI. Os genes encontrados no capítulo 4, no geral, desempenham um papel no metabolismo muscular e lipídico. Para ambas as características, alguns DAS pertenciam às famílias de genes da miosina e miotilina. Os resultados dessa tese indicam possíveis genes candidatos e fornecem a base teórica para uma melhor compreensão dos mecanismos moleculares do desenvolvimento e cresimento da AOL, bem como da deposição de GI em bovinos da raça Nelore.
RNA sequencing (RNA-Seq) is an approach for screening the expression of functional candidate genes and identifying important molecular mechanisms creating variation in pathways that result in different tissue phenotypes. Since ribeye muscle area (REA) and intramuscular fat content (IF) are polygenic traits, many genes and mechanisms that induce differences in muscle growth and development, as well as IF content of the Nelore cattle still unknown. The aim was to study the muscle transcriptome of the Nelore cattle with the purpose of identifying differentially expressed genes (DEGs) and differentially expressed alternative splicing events (DAS) associated with the carcass (REA) and meat (IF) traits, through of the RNA-seq approach. Therefore, a total of 80 animals were sequenced and phenotyped for REA and IF contet (measured by the chemical method - which evaluate total lipids). The results were presented in chapters 2, 3 and 4. In chapter 2, 15 animals with highest REA and 15 animals with lowest REA were selected for differential expression analysis. Upon analysis, 289 DEGs were identified (q-value ≤0.05): 183 upregulated and 106 downregulated in the highest REA group. Genes belonging to important families, such as actin, myosin, collagen, integrin, solute carrier, ubiquitin, and kelch-like, were among the DEGs. Gene set enrichment analysis showed that many of the significantly enriched gene ontology (GO) terms are closely associated with muscle development, growth, and degradation. Through co-expression network analysis, we predicted three hub genes (PPP3R1, FAM129B and UBE2G1). These genes are involved in muscle growth and proteolysis. In chapter 3, 15 animals with highest and 15 animals with lowest IF content were selected for differential expression analysis. Upon analysis, 65 DEGs were identified, including 21 upregulated and 44 downregulated genes in highest IF content animals. Gene set enrichment analysis was performed and showed that the GO terms were closely associated with lipid and muscle metabolism, as well as genes related to the immune system. Gene co-expression network analysis showed three hub genes (PDE4D, KLHL30, and IL1RAP) that may play a role in cellular and/or systemic lipid biology in Nelore cattle. In Chapter 4, differentially expressed alternative splicing events (DAS), and their associated genes, were identified in the same groups of animals selected in Chapter 2 (phenotypically divergent for REA) and Chapter 3 (phenotypically divergent for IF content). The DAS events were identified from the exon-centric approach. The results showed 166 and 269 DAS events (p-value ≤ 0,05), respectively, for REA and IF. The exon cassette and alternative 3' splice site were the most frequently found events for both traits. The DAS found were transcribed for 125 and 219 genes, respectively for REA and for IF. The genes found in chapter 4, in general, play a role in muscle metabolism and systemic lipid biology. The results reported in this tese show possible candidate genes and provide a theoretical basis for a better understanding of the molecular mechanism of REA development and growth as well as the deposition of IF in Nelore cattle.
RNA sequencing (RNA-Seq) is an approach for screening the expression of functional candidate genes and identifying important molecular mechanisms creating variation in pathways that result in different tissue phenotypes. Since ribeye muscle area (REA) and intramuscular fat content (IF) are polygenic traits, many genes and mechanisms that induce differences in muscle growth and development, as well as IF content of the Nelore cattle still unknown. The aim was to study the muscle transcriptome of the Nelore cattle with the purpose of identifying differentially expressed genes (DEGs) and differentially expressed alternative splicing events (DAS) associated with the carcass (REA) and meat (IF) traits, through of the RNA-seq approach. Therefore, a total of 80 animals were sequenced and phenotyped for REA and IF contet (measured by the chemical method - which evaluate total lipids). The results were presented in chapters 2, 3 and 4. In chapter 2, 15 animals with highest REA and 15 animals with lowest REA were selected for differential expression analysis. Upon analysis, 289 DEGs were identified (q-value ≤0.05): 183 upregulated and 106 downregulated in the highest REA group. Genes belonging to important families, such as actin, myosin, collagen, integrin, solute carrier, ubiquitin, and kelch-like, were among the DEGs. Gene set enrichment analysis showed that many of the significantly enriched gene ontology (GO) terms are closely associated with muscle development, growth, and degradation. Through co-expression network analysis, we predicted three hub genes (PPP3R1, FAM129B and UBE2G1). These genes are involved in muscle growth and proteolysis. In chapter 3, 15 animals with highest and 15 animals with lowest IF content were selected for differential expression analysis. Upon analysis, 65 DEGs were identified, including 21 upregulated and 44 downregulated genes in highest IF content animals. Gene set enrichment analysis was performed and showed that the GO terms were closely associated with lipid and muscle metabolism, as well as genes related to the immune system. Gene co-expression network analysis showed three hub genes (PDE4D, KLHL30, and IL1RAP) that may play a role in cellular and/or systemic lipid biology in Nelore cattle. In Chapter 4, differentially expressed alternative splicing events (DAS), and their associated genes, were identified in the same groups of animals selected in Chapter 2 (phenotypically divergent for REA) and Chapter 3 (phenotypically divergent for IF content). The DAS events were identified from the exon-centric approach. The results showed 166 and 269 DAS events (p-value ≤ 0,05), respectively, for REA and IF. The exon cassette and alternative 3' splice site were the most frequently found events for both traits. The DAS found were transcribed for 125 and 219 genes, respectively for REA and for IF. The genes found in chapter 4, in general, play a role in muscle metabolism and systemic lipid biology. The results reported in this tese show possible candidate genes and provide a theoretical basis for a better understanding of the molecular mechanism of REA development and growth as well as the deposition of IF in Nelore cattle.
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Palavras-chave
AOL, Bovino, Genes, Gordura intramuscular, Splicing alternativo, REA, Bovine, Intramuscular fat, Alternative splicing