Differential display of expressed genes reveals a novel function of SFRS18 in regulation of intramuscular fat deposition.
ABSTRACT: Intramuscular fat (IMF) content plays a key role in establishing pork quality. In the present study, differential-display reverse transcription-polymerase chain reaction (DDRT-PCR) was used to identify differentially expressed (DE) genes between longissimus dorsi (LD) muscles with extremely different IMF content. A major DE gene associated with IMF content was identified as splicing factor serine-arginine rich protein (SFRS18) gene, also known as SRrp130. The gene exhibited relatively higher expression levels in LD muscles with higher IMF content. A full-length cDNA sequence of pig SFRS18 gene was obtained by in silico comparative cloning coupled with PCR target sequencing, while the current EST (expressed sequence tag) database supported two transcript variants of the pig gene. Differential expression of the SFRS18 gene was further confirmed using quantitative PCR. The mRNA levels of SFRS18 gene showed significant and positive correlation with IMF content in LD muscle (r = 0.54, P < 0.01). Collectively, these results suggest that the SFRS18 gene is involved in the regulation of IMF deposition in pig and that it may be a useful tool in selecting animals for desired amounts of fatness for high quality pork.
Project description:Intramuscular fat (IMF) is an important economic trait for pork quality and a complex quantitative trait regulated by multiple genes. The objective of this work was to investigate the novel transcriptional effects of a multigene pathway on IMF deposition in the longissimus dorsi (LD) muscles of pigs. Potential signaling pathways were screened by mining data from three gene expression profiles in the Gene Expression Omnibus (GEO) database. We designed quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) arrays for the candidate signaling pathways to verify the results in the LD muscles of two pig breeds with different IMF contents (Large White and Min). Western blot analysis was used to detect the expression levels of several candidate proteins. Our results showed that the AMPK signaling pathway was screened via bioinformatics analysis. Ten key hub genes of this signaling pathway (AMPK, ADIPOR1, ADIPOR2, LKB1, CAMKKβ, CPT1A, CPT1B, PGC-1α, CD36, and ACC1) were differentially expressed between the Large White and Min pigs. Western blot analysis further confirmed that LKB1/CaMKK2-AMPK-ACC1-CPT1A axis dominates the activity of AMPK signaling pathway. Statistical analyses revealed that AMPK signaling pathway activity clearly varied among the two pig breeds. Based on these results, we concluded that the activation of the AMPK signaling pathway plays a positive role in reducing IMF deposition in pigs.
Project description:The intramuscular fat content (IMF) refers to the amount of fat within muscles, including the sum of phospholipids mainly found in cell membranes, triglycerides and cholesterol, and is determined both by hyperplasia and hypertrophy of adipocyte during the development of pigs. The IMF content is an important economic trait that is genetically controlled by multiple genes. The Laiwu pig is an indigenous fatty pig breed distributed in North China, characterized by excessively higher level of IMF content (9%~12%), therefore, is suitable for the identification of genes controlling IMF variations. To identify genes underlying IMF deposition, we performed genome-wide transcriptome and methylome analyses on longissimus dorsi (LD) muscle in Laiwu pigs across four developmental stages.A total of 22,524 expressed genes were detected and 1158 differentially expressed genes (DEGs) were hierarchically clustered in the LD muscle over four developmental stages from 60 d to 400 d. These genes were significantly clustered into four temporal expression profiles, and genes participating in fat cell differentiation and lipid biosynthesis processes were identified. From 120 d to 240 d, the period with the maximum IMF deposition rate, the lipid biosynthesis related genes (FOSL1, FAM213B and G0S2), transcription factors (TFs) (EGR1, KLF5, SREBF2, TP53 and TWIST1) and enriched pathways (steroid biosynthesis and fatty acid biosynthesis) were revealed; and fat biosynthesis relevant genes showing differences in DNA methylation in gene body or intergenic region were detected, such as FASN, PVALB, ID2, SH3PXD2B and EGR1.This study provides a comprehensive landscape of transcriptome of the LD muscle in Laiwu pigs ranging from 60 to 400 days old, and methylome of the LD muscle in 120 d and 240 d Laiwu pigs. A set of candidate genes and TFs involved in fat biosynthesis process were identified, which were probably responsible for IMF deposition. The results from this study would provide a reference for the identification of genes controlling IMF variation, and for exploring molecular mechanisms underlying IMF deposition in pigs.
Project description:<h4>Objective</h4>Intramuscular fat (IMF) content plays an important role in meat quality. Identification of single nucleotide polymorphisms (SNPs) and genes related to pig IMF, especially using pig populations with high IMF content variation, can help to establish novel molecular breeding tools for optimizing IMF in pork and unveil the mechanisms that underlie fat metabolism.<h4>Methods</h4>We collected muscle samples of 453 Chinese Lulai black pigs, measured IMF content by Soxhlet petroleum-ether extraction method, and genotyped genome-wide SNPs using GeneSeek Genomic Profiler Porcine HD BeadChip. Then a genome-wide association study was performed using a linear mixed model implemented in the GEMMA software.<h4>Results</h4>A total of 43 SNPs were identified to be significantly associated with IMF content by the cutoff p<0.001. Among these significant SNPs, the greatest number of SNPs (n = 19) were detected on Chr.9, and two linkage disequilibrium blocks were formed among them. Additionally, 17 significant SNPs are mapped to previously reported quantitative trait loci (QTLs) of IMF and confirmed previous QTLs studies. Forty-two annotated genes centering these significant SNPs were obtained from Ensembl database. Overrepresentation test of pathways and gene ontology (GO) terms revealed some enriched reactome pathways and GO terms, which mainly involved regulation of basic material transport, energy metabolic process and signaling pathway.<h4>Conclusion</h4>These findings improve our understanding of the genetic architecture of IMF content in pork and facilitate the follow-up study of fine-mapping genes that influence fat deposition in muscle.
Project description:In recent years, pig producers have struggled with the problem of low intramuscular fat levels in pork, which impacts palatability and ultimately meat quality. Reduced levels of intramuscular fat are likely the result of breeding objectives aimed at increasing lean meat content. In this study, three mutations within candidate genes for fat content (SCD, ACACA, and FASN) were selected, based on RNA-seq results and the relationship between polymorphisms in genes related to lipid metabolism, fattening and slaughter characteristics, as well as pork quality, including IMF level, were evaluated to identify selection markers. Moreover, their impact on gene expression was also examined. The PCR-RFLP (polymerase cha- in reaction - restriction fragments length) method was used to establish genotypes and effect sizes of potential genetic markers were estimated using a GLM model. It was identified that a FASN missense variant was positively associated with the expression level of this gene, which suggested its linkage with a mutation having a regulatory function. The association study indicated that the FASN missense variant may play a role in the determination of feed conversion and meat colour. In turn, a mutation in the ACACA gene showed a relationship with IMF content in the Pu?awska breed where the differences reached as much as 20%. We suggest considering all three mutations in further studies based on different pig populations due to the crucial role of SCD, ACACA, and FASN genes in lipid metabolism.
Project description:Intramuscular fat (IMF) content has been generally recognized as a desirable trait in pork meat because of its positive effect on eating quality. An effective approach to enhance IMF content in pork is the generation of transgenic pigs. In this study, we used somatic cell nuclear transfer (SCNT) to generate cloned pigs exhibiting ectopic expression of phosphoenolpyruvate carboxykinase (PEPCK-C) driven by an ?-skeletal-actin gene promoter, which was specifically expressed in skeletal muscle. Using qRT-PCR and Western blot analysis, we demonstrated that PEPCK-C was functionally expressed and had a significant effect on total fatty acid content in the skeletal muscle of the transgenic pigs, while the n-6/n-3 polyunsaturated fatty acid (PUFA) ratio showed no difference between transgenic and control pigs. Thus, genetically engineered PEPCK-C<sup>mus</sup> pigs may be an effective solution for the production of IMF-enriched pork.
Project description:Intramuscular fat (IMF) content is an important trait that can affect pork quality. Previous studies have identified many genes that can regulate IMF. Long intergenic non-coding RNAs (lincRNAs) are emerging as key regulators in various biological processes. However, lincRNAs related to IMF in pig are largely unknown, and the mechanisms by which they regulate IMF are yet to be elucidated. Here we reconstructed 105,687 transcripts and identified 1,032 lincRNAs in pig longissimus dorsi muscle (LDM) of four stages with different IMF contents based on published RNA-seq. These lincRNAs show typical characteristics such as shorter length and lower expression compared with protein-coding genes. Combined with methylation data, we found that both the promoter and genebody methylation of lincRNAs can negatively regulate lincRNA expression. We found that lincRNAs exhibit high correlation with their protein-coding neighbors in expression. Co-expression network analysis resulted in eight stage-specific modules, gene ontology and pathway analysis of them suggested that some lincRNAs were involved in IMF-related processes, such as fatty acid metabolism and peroxisome proliferator-activated receptor signaling pathway. Furthermore, we identified hub lincRNAs and found six of them may play important roles in IMF development. This work detailed some lincRNAs which may affect of IMF development in pig, and facilitated future research on these lincRNAs and molecular assisted breeding for pig.
Project description:BACKGROUND: Intramuscular fat (IMF) content is positively correlated with aspects of pork palatability, including flavour, juiciness and overall acceptability. The ratio of energy to protein in the finishing diet of growing pigs can impact on IMF content with consequences for pork quality. The objective of this study was to compare gene expression profiles of Musculus semimembranosus (SM) of animals divergent for IMF as a consequence of protein dietary restriction in an isocaloric diet. The animal model was derived through the imposition of low or high protein diets during the finisher stage in Duroc gilts. RNA was extracted from post mortem SM tissue, processed and hybridised to Affymetrix porcine GeneChip® arrays. RESULTS: IMF content of SM muscle was increased on the low protein diet (3.60?±?0.38% versus 1.92?±?0.35%). Backfat depth was also greater in animals on the low protein diet, and average daily gain and feed conversion ratio were lower, but muscle depth, protein content and moisture content were not affected. A total of 542 annotated genes were differentially expressed (DE) between animals on low and high protein diets, with 351 down-regulated and 191 up-regulated on the low protein diet. Transcript differences were validated for a subset of DE genes by qPCR. Alterations in functions related to cell cycle, muscle growth, extracellular matrix organisation, collagen development, lipogenesis and lipolysis, were observed. Expression of adipokines including LEP, TNF? and HIF1? were increased and the hypoxic stress response was induced. Many of the identified transcriptomic responses have also been observed in genetic and fetal programming models of differential IMF accumulation, indicating they may be robust biological indicators of IMF content. CONCLUSION: An extensive perturbation of overall energy metabolism in muscle occurs in response to protein restriction. A low protein diet can modulate IMF content of the SM by altering gene pathways involved in lipid biosynthesis and degradation; however this nutritional challenge negatively impacts protein synthesis pathways, with potential consequences for growth.
Project description:Intramuscular fat (IMF) content is closely related to various meat traits, such as tenderness, juiciness, and flavor. The IMF content varies considerably among pig breeds with different genetic backgrounds. Long intergenic non-coding RNAs (lincRNAs) have been widely identified in many species and found to be an important class of regulators that can participate in multiple biological processes. However, the mechanism behind lincRNAs regulation of pig IMF content remains unknown and requires further study. In our study, we identified a total of 156 lincRNAs in the longissimus dorsi muscle of Wei (fat-type) and Yorkshire (lean-type) pigs using previously published data. These identified lincRNAs have shorter transcript length, longer exon length, lower exon number, and lower expression level as compared with protein-coding transcripts. We predicted potential target genes (PTGs) that are potentially regulated by lincRNAs in cis or trans regulation. Gene ontology and pathway analyses indicated that many potential lincRNAs target genes are involved in IMF-related processes or pathways, such as fatty acid catabolic process and adipocytokine signaling pathway. In addition, we analyzed quantitative trait locus (QTL) sites that differentially expressed lincRNAs (DE lincRNAs) between Wei and Yorkshire pigs co-localized. The QTL sites where DE lincRNAs co-localize are mostly related to IMF content. Furthermore, we constructed a co-expressed network between DE lincRNAs and their differentially expressed PTGs (DEPTGs). On the basis of their expression levels, we suggest that many DE lincRNAs can affect IMF development by positively or negatively regulating their PTGs. This study identified and analyzed some lincRNAs- and PTGs-related IMF development of the two pig breeds and provided new insight into research on the roles of lincRNAs in the two types of breeds.
Project description:This study aims to determine the polymorphism and mRNA expression pattern of the heart-type fatty acid-binding protein (H-FABP) gene and their association with intramuscular fat (IMF) content in the breast and leg muscles of Baicheng oil chicken (BOC). A total of 720 chickens, including 240 black Baicheng oil chicken (BBOC), 240 silky Baicheng oil chicken (SBOC), and 240 white Baicheng oil chicken (WBOC) were raised. Three genotypes of H-FABP gene second extron following AA, AB, and BB were detected by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) strategy. The G939A site created AA genotype and G956A site created BB genotype. The content of IMF in AA genotype in breast muscle of BBOC was significantly higher than that of AB (p = 0.0176) and the genotype in leg muscle of WBOC was significantly higher than that of AB (p = 0.0145). The G939A site could be taken as genetic marker for higher IMF content selecting for breast muscle of BBOC and leg muscle of WBOC. The relative mRNA expression of H-FABP was measured by real-time PCR at 30, 60, 90, and 120 d. The IMF content significantly increased with age in both muscles. The mRNA expression level of H-FABP significantly decreased with age in both muscles of the three types of chickens. Moreover, a significant negative correlation between H-FABP abundance and IMF content in the leg muscles of WBOC (p = 0.035) was observed. The mRNA expression of H-FABP negatively correlated with the IMF content in both breast and leg muscles of BOC sat slaughter time.
Project description:Iberian pig production includes purebred (IB) and Duroc-crossbred (IBxDU) pigs, which show important differences in growth, fattening and tissue composition. This experiment was conducted to investigate the effects of genetic type and muscle (Longissimus dorsi (LD) vs Biceps femoris (BF)) on gene expression and transcriptional regulation at two developmental stages. Nine IB and 10 IBxDU piglets were slaughtered at birth, and seven IB and 10 IBxDU at four months of age (growing period). Carcass traits and LD intramuscular fat (IMF) content were measured. Muscle transcriptome was analyzed on LD samples with RNA-Seq technology. Carcasses were smaller in IB than in IBxDU neonates (p < 0.001), while growing IB pigs showed greater IMF content (p < 0.05). Gene expression was affected (p < 0.01 and Fold change > 1.5) by the developmental stage (5,812 genes), muscle type (135 genes), and genetic type (261 genes at birth and 113 at growth). Newborns transcriptome reflected a highly proliferative developmental stage, while older pigs showed upregulation of catabolic and muscle functioning processes. Regarding the genetic type effect, IBxDU newborns showed enrichment of gene pathways involved in muscle growth, in agreement with the higher prenatal growth observed in these pigs. However, IB growing pigs showed enrichment of pathways involved in protein deposition and cellular growth, supporting the compensatory gain experienced by IB pigs during this period. Moreover, newborn and growing IB pigs showed more active glucose and lipid metabolism than IBxDU pigs. Moreover, LD muscle seems to have more active muscular and cell growth, while BF points towards lipid metabolism and fat deposition. Several regulators controlling transcriptome changes in both genotypes were identified across muscles and ages (SIM1, PVALB, MEFs, TCF7L2 or FOXO1), being strong candidate genes to drive expression and thus, phenotypic differences between IB and IBxDU pigs. Many of the identified regulators were known to be involved in muscle and adipose tissues development, but others not previously associated with pig muscle growth were also identified, as PVALB, KLF1 or IRF2. The present study discloses potential molecular mechanisms underlying phenotypic differences observed between IB and IBxDU pigs and highlights candidate genes implicated in these molecular mechanisms.