RESUMO
Research on gene regulation has been made possible with the help of RNA sequencing applications such as RNA-Seq technology for high-throughput sequencing platforms. Recent studies have explored the transcriptomes from different tissues of domestic animals using RNA-Seq technology, but little research has been done to study the transcriptomes of breeds of sheep having different adipose tissue deposition mechanisms, such as Mongolian and Lanzhou fat-tailed sheep. In this study, Mongolian and Lanzhou fat-tailed sheep were selected as experimental breeds, and six libraries (three libraries per breed) were constructed. A total of 286 Mb of high-quality reads was obtained, and three-quarters of the reads were mapped to the reference genome per library. In addition, there were 16,257, 16,186, 16,254, 16,827, 16,437, and 15,761 known reference genes in the six constructed libraries (LCL1, LCL2, LCL3, MCL1, MCL2, and MCL3, respectively). Seven genes were differentially expressed: four were upregulated and three were downregulated in liver tissue between the MCL and LCL groups; 65,303, 65,442, 63,426, 76,267, 69,853, and 57,439 potential cSNPs were detected in the six libraries, respectively, with the G/R substitution occurring most commonly. There were 24,239, 22,283, 22,457, 26,635, 27,093, and 18,700 alternate splicing (AS) events in the six libraries. Intron retention was the most common AS event, followed by alternative 3' splice sites. These results indicate that there are many differences in the liver transcriptomes of Mongolian and Lanzhou fat-tailed sheep breeds. Such results may provide fundamental information for further research on defining the sheep genome.
Assuntos
Fígado/metabolismo , Análise de Sequência de RNA/métodos , Carneiro Doméstico/genética , Transcriptoma/genética , Processamento Alternativo/genética , Animais , Cruzamento , Perfilação da Expressão Gênica , Humanos , Íntrons/genéticaRESUMO
To evaluate stearoyl-CoA desaturase (SCD), hormone-sensitive lipase (HSL), lipoprotein lipase (LPL), and peroxisome proliferator-activated receptor (PPARγ) expression in Lanzhou fat-tailed sheep (with and without docked tails), 18 rams were randomly divided into two equal groups (docked group, LT; control group, LC). These data were also used to increase the understanding of sheep fat deposition and metabolism. All animals were harvested at the age of 18 months, and expression was determined for 10 tissues. The results indicated that the fat weight of each tissue in LT was higher than in LC (P < 0.05). SCD expression in semitendinosus, omentum majus fat (OF), subcutaneous fat, kidney fat (KF), and subcutaneous rump fat was higher in LT than in LC rams (P < 0.05). Trends (P < 0.10) associated with higher HSL expression of LC in comparison to that of LT rams in intestinal fat, OF, and KF tissues were detected. Numerically, LPL expression was the highest in KF, OF, and kidney tissues, but there were few differences (P > 0.10). PPARγexpression was greater in LT than in LC rams in liver tissues (P < 0.05), but there were few differences in other tissues. No significant differences were found with regard to the regression analysis of expression and adipose tissue weights, but the two indices exhibited the same trend. The results indicated that changes in fatty deposits may be due to the common control of docking management and the minor effects associated with the regulation of SCD, HSL, LPL, and PPARγexpression.
Assuntos
Metabolismo dos Lipídeos/fisiologia , Cauda/cirurgia , Tecido Adiposo/metabolismo , Amputação Cirúrgica/veterinária , Animais , Ácidos Graxos/metabolismo , Lipase Lipoproteica/metabolismo , Masculino , PPAR gama/metabolismo , Ovinos , Carneiro Doméstico , Estearoil-CoA Dessaturase/metabolismoRESUMO
ASB15 is a member of the ankyrin repeat and suppressor of cytokine signaling box family, and is predominantly expressed in skeletal muscle. In the present study, an F2 resource population of Gushi chickens crossed with Anka broilers was used to investigate the genetic effects of the chicken ASB15 gene. Two single nucleotide polymorphisms (SNPs) (rs315759231 A>G and rs312619270 T>C) were identified in exon 7 of the ASB15 gene using forced chain reaction-restriction fragment length polymorphism and DNA sequencing. One was a missense SNP (rs315759231 A>G) and the other was a synonymous SNP (rs312619270 T>C). The rs315759231 A>G polymorphism was significantly associated with body weight at birth, 12-week body slanting length, semi-evisceration weight, evisceration weight, leg muscle weight, and carcass weight (P < 0.05). The rs312619270 T>C polymorphism was significantly associated with body weight at birth, 4, 8, and 12-week body weight, 8-week shank length, 12-week breast bone length, 8 and 12-week body slanting length, breast muscle weight, and carcass weight (P < 0.05). Our results suggest that the ASB15 gene profoundly affects chicken growth and carcass traits.
Assuntos
Proteínas Aviárias/genética , Galinhas/crescimento & desenvolvimento , Galinhas/genética , Estudos de Associação Genética , Carne , Polimorfismo de Nucleotídeo Único/genética , Animais , Sequência de Bases , Eletroforese em Gel de Ágar , Feminino , Frequência do Gene/genética , Técnicas de Genotipagem , Desequilíbrio de Ligação/genética , Masculino , Dados de Sequência Molecular , Polimorfismo de Fragmento de Restrição , Característica Quantitativa Herdável , Análise de Sequência de DNARESUMO
Lambing performance of sheep is the most important economic trait and is regarded as a critic factoring affecting the productivity in sheep industry. Ovary plays the most roles in lambing trait. To establish the optimum two-dimensional electrophoresis system (2-DE) of ovine ovarian tissue, the common protein extraction methods of animal tissue (trichloroacetic acid/acetone precipitation and direct schizolysis methods) were used to extract ovine ovarian protein, and 17-cm nonlinear immobilized PH 3-10 gradient strips were used for 2-DE. The sample handling, loading quantity of the protein sample, and isoelectric focusing (IEF) steps were manipulated and optimized in this study. The results indicate that the direct schizolysis III method, a 200-µg loading quantity of the protein sample, and IEF steps II (20°C active hydration, 14 hâ500 V, 1 hâ1000 V 1 hâ1000-9000 V, 6 hâ80,000 VHâ500 V 24 h) are optimal for 2-DE analysis of ovine ovarian tissue. Therefore, ovine ovarian tissue proteomics 2-DE was preliminarily established by the optimized conditions in this study; meanwhile, the conditions identified herein could provide a reference for ovarian sample preparation and 2-DE using tissues from other animals.
Assuntos
Eletroforese em Gel Bidimensional/métodos , Ovário/metabolismo , Proteoma/análise , Proteômica/métodos , Acetona/química , Animais , Precipitação Química , Feminino , Concentração de Íons de Hidrogênio , Focalização Isoelétrica , Proteoma/isolamento & purificação , Reprodutibilidade dos Testes , Ovinos , Ácido Tricloroacético/químicaRESUMO
The growth hormone gene plays an important role in the physiological function of an organism. The current study aimed to investigate the correlation between polymorphisms in the 5' regulatory region, exon 4, and 3' untranslated region (UTR) of the sheep GH gene and sheep growth traits. The DNA from 510 adult sheep was analyzed by DNA sequencing and polymerase chain reaction single-strand conformation polymorphism. Two alleles (A and B) and 3 genotypes (AA, AB, and BB), 2 alleles (A and B) and 3 genotypes (AA, AB, and BB), and 3 alleles (A, B, and C) and 4 genotypes (AA, AB, BB, and AC) were found within the 5' regulatory region, exon 4, and 3' UTR, respectively. In Tibetan sheep, the association analysis indicated that there were statistically significant differences in the scores of weight, length, and heart girth within the 5' regulatory region; weight, length, wither height, and heart girth within exon 4; and weight, length, wither height, and heart girth within the 3' UTR among the different genotypes. For exon 4, Poll Dorset sheep individuals with genotype AA showed a lower score than those of genotypes BB and AB (P<0.05). With regard to the 3' UTR, Poll Dorset sheep with genotype AC showed higher scores than those of genotypes AA and AB (P<0.05).
Assuntos
Hormônio do Crescimento/genética , Polimorfismo Genético , Característica Quantitativa Herdável , Ovinos/crescimento & desenvolvimento , Ovinos/genética , Regiões 3' não Traduzidas , Alelos , Animais , Éxons , Frequência do Gene , Estudos de Associação Genética , Genótipo , Dados de Sequência Molecular , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNARESUMO
Current studies find that degenerated cartilage endplates (CEP) of vertebrae, with fewer diffusion areas, decrease nutrient supply and accelerate intervertebral disc degeneration. Many more apoptotic cells have been identified in degenerated than in normal endplates, and may be responsible for the degenerated grade. Previous findings suggest that inhibition of apoptosis is one possible approach to improve disc regeneration. It is postulated that inhibition of CEP cell apoptosis may be responsible for the regeneration of endplates. Caspase-3, involved in the execution phase of apoptosis, is a candidate for regulating the apoptotic process. In the present study, CEP cells were incubated in 1% fetal bovine serum. Activated caspases were detected to identify the apoptotic pathway, and apoptosis was quantified by flow cytometry. Lentiviral caspase-3 short hairpin RNA (shRNA) was employed to study its protective effects against serum deprivation. Silencing of caspase-3 expression was quantified by reverse transcription-polymerase chain reaction and Western blots, and inhibition of apoptosis was quantified by flow cytometry. Serum deprivation increased apoptosis of rat CEP cells through activation of a caspase cascade. Lentiviral caspase-3 shRNA was successfully transduced into CEP cells, and specifically silenced endogenous caspase-3 expression. Surviving cells were protected by the downregulation of caspase-3 expression and activation. Thus, lentiviral caspase-3 shRNA-mediated RNAi successfully silenced endogenous caspase-3 expression, preventing inappropriate or premature apoptosis.
Assuntos
Apoptose/fisiologia , Caspase 3/metabolismo , Condrócitos/metabolismo , Lentivirus/genética , Interferência de RNA/fisiologia , Inanição/metabolismo , Animais , Anexina A5 , Western Blotting , Cartilagem/metabolismo , Caspase 8/metabolismo , Caspase 9/metabolismo , Bovinos , Citometria de Fluxo , Vetores Genéticos/metabolismo , Microscopia de Fluorescência , Cultura Primária de Células , Propídio , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Soro/fisiologia , TransfecçãoRESUMO
Current studies find that degenerated cartilage endplates (CEP) of vertebrae, with fewer diffusion areas, decrease nutrient supply and accelerate intervertebral disc degeneration. Many more apoptotic cells have been identified in degenerated than in normal endplates, and may be responsible for the degenerated grade. Previous findings suggest that inhibition of apoptosis is one possible approach to improve disc regeneration. It is postulated that inhibition of CEP cell apoptosis may be responsible for the regeneration of endplates. Caspase-3, involved in the execution phase of apoptosis, is a candidate for regulating the apoptotic process. In the present study, CEP cells were incubated in 1% fetal bovine serum. Activated caspases were detected to identify the apoptotic pathway, and apoptosis was quantified by flow cytometry. Lentiviral caspase-3 short hairpin RNA (shRNA) was employed to study its protective effects against serum deprivation. Silencing of caspase-3 expression was quantified by reverse transcription-polymerase chain reaction and Western blots, and inhibition of apoptosis was quantified by flow cytometry. Serum deprivation increased apoptosis of rat CEP cells through activation of a caspase cascade. Lentiviral caspase-3 shRNA was successfully transduced into CEP cells, and specifically silenced endogenous caspase-3 expression. Surviving cells were protected by the downregulation of caspase-3 expression and activation. Thus, lentiviral caspase-3 shRNA-mediated RNAi successfully silenced endogenous caspase-3 expression, preventing inappropriate or premature apoptosis.
Assuntos
Animais , Bovinos , Apoptose/fisiologia , /metabolismo , Condrócitos/metabolismo , Lentivirus/genética , Interferência de RNA/fisiologia , Inanição/metabolismo , Western Blotting , Cartilagem/metabolismo , Caspase 9/metabolismo , /metabolismo , Citometria de Fluxo , Vetores Genéticos/metabolismo , Microscopia de Fluorescência , Cultura Primária de Células , Propídio , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Soro/fisiologia , TransfecçãoRESUMO
Thyroid transcription factor-1 (TTF-1), a member of the Nkx2 family of homeodomain-containing proteins, is involved in binding to and in activating the promoters of several important genes in the thyroid, lungs, and brain, and in regulating expression of these tissue-specific genes. We investigated potential roles of sheep (Ovis aries) TTF-1 in regulating cell fate and organ morphogenesis and in controlling puberty and reproductive capability of females. We amplified and cloned the sheep TTF-1 full-length DNA for the first time, analyzed its functional domains and regions, predicted molecular structure of its homeodomain and DNA-binding sites, and examined its expression in pituitary, brain, thyroid gland, ovary, and hypothalamus. We found that sheep TTF-1 has a high degree of homologous identity with that of other mammals, and it has several important domains including domain N, DNA-binding domain, domain C, TN-domain, domain I, and NK2-SD. The DNA-binding domain of sheep TTF-1 has 10 potential DNA-binding sites and is a novel mammalian homeodomain that shows considerable sequence homology with the corresponding rat homeodomain. Several functional regions in sheep TTF-1 share high sequence identity with rat TTF-1, indicating that these regions may have the same activity as in the rat. Expression of TTF-1 in several specific tissues implies that sheep TTF-1 in involved in sheep sexual development and reproductive capability. These results suggest a role of sheep TTF-1 in enhancing sheep reproduction performance and we propose it as a candidate gene for selection.