Estudo de proteínas diferencialmente expressas e integração biológica multi ômicas para características de qualidade de carne e carcaça em bovinos da raça Nelore
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2023-04-05
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Universidade Estadual Paulista (Unesp)
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As técnicas ômicas são ferramentas poderosas que permitem o estudo abrangente e sistemático dos componentes biológicos em um organismo. Isso inclui a análise do DNA, RNA e proteínas, que fornecem informações sobre diferentes níveis de organização molecular. No entanto, apesar dos avanços obtidos pelas técnicas isoladamente, é cada vez mais aceito pela comunidade científica a ideia de que nenhuma delas é capaz de lidar com a complexidade dos sistemas biológicos por si só. Dessa forma, uma abordagem integrativa por meio de análises de enriquecimento funcional de dados multi-ômicos, pode fornecer informações mais abrangentes e precisas sobre genes e redes biológicas envolvidos em fenótipos de interesse. Portanto, o objetivo do trabalho foi a identificação de biomarcadores por meio de análise proteômica, além de uma abordagem biológica integrativa combinando os dados de GWAS (associação genômica ampla), transcriptômicos (RNA-Seq) e proteômicos (LC-MS/MS) a fim de identificar os principais loci e vias associados às características de interesse. Para isso, 24 bovinos Nelore extremos para cada característica estudada (maciez, área de olho de lombo (AOL), marmoreio e espessura de gordura subcutânea (EGS) foram sequenciados e fenotipados. Os resultados foram apresentados no capítulo 2 e 3. No capítulo 2, foram selecionados para análise proteômica, 12 animais com maiores e 12 animais com menores fenótipos para maciez, AOL, marmoreio e EGS. A análise proteômica foi realizada utilizando as contagens espectrais brutas dos mesmos grupos extremos, por meio do pacote em R msmsTest. Proteínas envolvidas na organização do citoesqueleto, proteólise e pertencentes a família de pequenas proteínas de choque térmico foram identificadas como diferencialmente expressas para maciez da carne. Além disso foram identificadas proteínas centrais para a característica, incluindo CRYAB, FGG, PSMB5 e RDX. Para o marmoreio foram identificadas proteínas associadas a organização do citoesqueleto, ligação a actina, adipogênese e proliferação celular, além de proteínas centrais como ATP5F1A e TPI1. Considerando as AOL, as proteínas estavam associadas a proliferação celular, reparo do DNA, replicação, transcrição e proteínas pertencentes a família SERPINA. Além disso, a análise de co-expressão indicou a proteína DOCK7 como central para a característica. Para EGS as proteínas identificadas estavam envolvidas na regulação do citoesqueleto de actina, estabilização da matriz extracelular e estruturação celular. Além disso, a CAMK2B foi identificada como central para o fenótipo. No capítulo 4 os resultados das análises GWAS foram apresentadas com base na proporção de variância aditiva explicado por janelas de 1 Mb. Para as análises de RNA-Seq, foram utilizados os mesmos grupos de animais extremos considerados na análise proteômica. Estes conjuntos de 24 animais foram submetidos à análise de expressão diferencial por meio do software DESeq2. No geral, os genes e proteínas estavam associados ao crescimento, regulação do ciclo celular, estrutura celular e resposta ao estresse térmico (AUTS2, CAST RDX e PSMD7). Considerando a AOL, genes e proteínas estavam envolvidos com processos apoptóticos, desenvolvimento muscular e regulação do ciclo celular. Para a característica de marmoreio foram identificadas proteínas de ligação à actina, proteínas formadoras de microtúbulos e proteínas estruturais, além de genes envolvidos na composição e síntese de ácidos graxos a genes centrais como CAND1, ACTN4, FGFR2 e NCOR2. Para EGS foram identificados genes neuronais, além de transcritos e proteínas associados a organização citoesqueleto de actina e formação de microtúbulos. Além disso, foram identificados genes centrais relacionados com a ubiquitinação (CAND1), regulação do metabolismo energético (CAMK2D) e remodelação de tecidos (PAPPA). O presente estudo permitiu o conhecimento de possíveis genes e proteínas candidatos, além de fornecer uma melhor compreensão teórica dos processos biológicos associados a características de carne e carcaça. Estas informações podem ser úteis em futuras pesquisas com o objetivo de elucidar potenciais marcadores genéticos.
Omics techniques are powerful tools that allow for the comprehensive and systematic study of biological components in an organism. This includes the analysis of DNA, RNA, and proteins, which provide information about different levels of molecular organization. However, despite the advances made by individual techniques, it is increasingly accepted by the scientific community that none of them is capable of dealing with the complexity of biological systems on its own. Thus, an integrative approach through functional enrichment analyses of multi-omics data may provide more comprehensive and accurate information about genes and biological networks involved in phenotypes of interest. Therefore, the objective of this work was to identify biomarkers through proteomic analysis, in addition to an integrative biological approach combining data from genome-wide association studies (GWAS), transcriptomics (RNA-Seq), and proteomics (LC-MS/MS) to identify the main loci and pathways associated with the traits of interest. For this purpose, 24 Nellore cattle extremes for each studied trait (tenderness, rib eye area (REA), marbling, and subcutaneous fat thickness (SFT) were sequenced and phenotyped. The results were presented in chapters 2 and 3. In chapter 2, 12 animals with the highest and 12 animals with the lowest phenotypes for tenderness, REA, marbling, and SFT were selected for proteomic analysis. The proteomic analysis was performed using the raw spectral counts of the same extreme groups, through the R package msmsTest. Proteins involved in cytoskeleton organization, proteolysis, and belonging to the small heat shock protein family were identified as differentially expressed for meat tenderness. Additionally, central proteins for the characteristic, including CRYAB, FGG, PSMB5, and RDX were identified. For marbling, proteins associated with cytoskeleton organization, actin binding, adipogenesis, and cell proliferation were identified, as well as central proteins such as ATP5F1A and TPI1. Considering REA, the proteins were associated with cell proliferation, DNA repair, replication, transcription, and proteins belonging to the SERPINA family. Furthermore, the co-expression analysis indicated the protein DOCK7 as central for the characteristic. For SFT, the identified proteins were involved in actin cytoskeleton regulation, extracellular matrix stabilization, and cell structure. Additionally, CAMK2B was identified as central for the studied phenotype. In chapter 4, the results of GWAS analyses were presented based on the proportion of additive variance explained by 1 Mb windows. For RNA-Seq analyses, the same extreme animal groups considered in the proteomic analysis were used. These sets of 24 animals were subjected to differential expression analysis through the DESeq2 software. Overall, the genes and proteins were associated with growth, cell cycle regulation, cell structure, and response to heat stress (AUTS2, CAST, RDX, and PSMD7). Considering AOL, genes and proteins were involved in apoptotic processes, muscle development, and cell cycle regulation. For the marbling characteristic, actin-binding proteins, microtubule-forming proteins, and structural proteins, as well as genes involved in fatty acid composition and synthesis, such as CAND1, ACTN4, FGFR2, and NCOR2, were identified. For EGS, neuronal genes, as vii well as transcripts and proteins associated with actin cytoskeleton organization and microtubule formation, were identified. Additionally, central genes related to ubiquitination (CAND1), regulation of energy metabolism (CAMK2D), and tissue remodeling (PAPPA) were identified. This study allowed the identification of potential candidate genes and proteins, providing a better theoretical understanding of the biological processes associated with meat and carcass traits. This information can be useful in future research aiming to elucidate potential genetic markers.
Omics techniques are powerful tools that allow for the comprehensive and systematic study of biological components in an organism. This includes the analysis of DNA, RNA, and proteins, which provide information about different levels of molecular organization. However, despite the advances made by individual techniques, it is increasingly accepted by the scientific community that none of them is capable of dealing with the complexity of biological systems on its own. Thus, an integrative approach through functional enrichment analyses of multi-omics data may provide more comprehensive and accurate information about genes and biological networks involved in phenotypes of interest. Therefore, the objective of this work was to identify biomarkers through proteomic analysis, in addition to an integrative biological approach combining data from genome-wide association studies (GWAS), transcriptomics (RNA-Seq), and proteomics (LC-MS/MS) to identify the main loci and pathways associated with the traits of interest. For this purpose, 24 Nellore cattle extremes for each studied trait (tenderness, rib eye area (REA), marbling, and subcutaneous fat thickness (SFT) were sequenced and phenotyped. The results were presented in chapters 2 and 3. In chapter 2, 12 animals with the highest and 12 animals with the lowest phenotypes for tenderness, REA, marbling, and SFT were selected for proteomic analysis. The proteomic analysis was performed using the raw spectral counts of the same extreme groups, through the R package msmsTest. Proteins involved in cytoskeleton organization, proteolysis, and belonging to the small heat shock protein family were identified as differentially expressed for meat tenderness. Additionally, central proteins for the characteristic, including CRYAB, FGG, PSMB5, and RDX were identified. For marbling, proteins associated with cytoskeleton organization, actin binding, adipogenesis, and cell proliferation were identified, as well as central proteins such as ATP5F1A and TPI1. Considering REA, the proteins were associated with cell proliferation, DNA repair, replication, transcription, and proteins belonging to the SERPINA family. Furthermore, the co-expression analysis indicated the protein DOCK7 as central for the characteristic. For SFT, the identified proteins were involved in actin cytoskeleton regulation, extracellular matrix stabilization, and cell structure. Additionally, CAMK2B was identified as central for the studied phenotype. In chapter 4, the results of GWAS analyses were presented based on the proportion of additive variance explained by 1 Mb windows. For RNA-Seq analyses, the same extreme animal groups considered in the proteomic analysis were used. These sets of 24 animals were subjected to differential expression analysis through the DESeq2 software. Overall, the genes and proteins were associated with growth, cell cycle regulation, cell structure, and response to heat stress (AUTS2, CAST, RDX, and PSMD7). Considering AOL, genes and proteins were involved in apoptotic processes, muscle development, and cell cycle regulation. For the marbling characteristic, actin-binding proteins, microtubule-forming proteins, and structural proteins, as well as genes involved in fatty acid composition and synthesis, such as CAND1, ACTN4, FGFR2, and NCOR2, were identified. For EGS, neuronal genes, as vii well as transcripts and proteins associated with actin cytoskeleton organization and microtubule formation, were identified. Additionally, central genes related to ubiquitination (CAND1), regulation of energy metabolism (CAMK2D), and tissue remodeling (PAPPA) were identified. This study allowed the identification of potential candidate genes and proteins, providing a better theoretical understanding of the biological processes associated with meat and carcass traits. This information can be useful in future research aiming to elucidate potential genetic markers.
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Palavras-chave
GWAS, LC-MS/MS, RNA-seq, Seleção genética, Genética molecular, Genética animal