Characterization of <Emphasis Type="Italic">methionine adenosyltransferase 2β</Emphasis> gene expression in skeletal muscle and subcutaneous adipose tissue from obese and lean pigs

Methionine adenosyltransferase (MAT) catalyzes the biosynthesis of S-adenosylmethionine. Two genes (MAT1A and MAT2A) encode for the catalytic subunit of MAT, while a third gene (MAT2β) encodes for a regulatory subunit (MAT II β) that regulates the activity of the MAT2A-encoded isoenzyme and intracellular S-adenosylmethionine levels. Our previous work identified MAT2β as a candidate gene for intramuscular fat (IMF) deposition in porcine skeletal muscle by microarray technology. Here, we cloned porcine MAT2β cDNA and compared its expression pattern in subcutaneous adipose tissue and skeletal muscle from obese (Rongchang Breed) and lean (Pig Improvement Company, PIC) pigs (n = 6). The porcine MAT2β cDNA was 1,800 bp long and encodes for 334 amino acids sharing high similarity with other species. MAT2β is expressed at a higher level in liver and duodenum, followed by the stomach, fat and longissinus dorsi muscle. As expected, both subcutaneous fat content and IMF content were higher in obese than in lean pigs (both P < 0.01). MAT2β mRNA abundance was lower in both subcutaneous adipose tissue and skeletal muscle in obese pigs compared with lean pigs (both P < 0.01). MAT II β protein content was lower in skeletal muscle in obese than in lean pigs (P < 0.05), whereas the opposite was observed in subcutaneous adipose tissue (P < 0.01). These data demonstrated an obesity-related expression variation of the MAT II β subunit in skeletal muscle and adipose tissue in pigs, and suggest a novel role for the MAT2β gene in regulation of IMF deposition in skeletal muscle.     

Molecular characterization of the porcine <Emphasis Type="Italic">JHDM1A</Emphasis> gene associated with average daily gain: evaluation its role in skeletal muscle development and growth

JHDM1A, a member of the JHDM (JmjC-domain-containing histone demethylase) family, plays an central role in gene silencing, cell cycle, cell growth and cancer development through histone H3K36 demethylation modification. Here reported the cloning, expression, chromosomal location and association analysis with growth traits of porcine JHDM1A gene. Sequence analysis showed that the porcine JHDM1A gene encodes 1,162 amino acids and contains JmjC, F-box, and CXXC zinc-finger domains, which coding sequence and deduced protein shares 91 and 99% similarity with human JHDM1A, respectively. Spatio-Temporal expression analysis indicated that the mRNA expression of porcine JHDM1A had significantly higher levels in the middle (65 days) and later (90 days) period’s embryo skeletal muscle than that of 33 days, and showed a ubiquitously expression but with the highest abundance in kidney, lung and liver of an adult pig. Radiation hybrid mapping and the following linkage mapping data indicate that JHDM1A maps to 2p17 region of pig chromosome 2 (SSC2). Allele frequency differences were detected in different pig breeds and an association study was performed with a SNP within 3′UTR. The results showed that there is a tendency for allele frequencies to differ between the fast growth breeds (Yorkshire) and slow growth pig breeds (Qingping pigs, Yushan Black pigs, Erhualian pigs and Dahuabai pigs). The association analysis using a Berkshire × Yorkshire F₂ population indicated that the C224G polymorphism had a highly significant association with average daily gain on test (P < 0.01), a trend association with average back fat thickness (P < 0.07), and significant associations (P < 0.01) on percent of average drip loss, Fiber Type II Ratio, muscle shear force and average lactate content in μmol/g. This study provides the first evidence that JHDM1A is differentially expressed in porcine embryonic skeletal muscle and associated with meat growth and quality traits.     

Molecular characterization and expression patterns of <Emphasis Type="Italic">Lbx1</Emphasis> in porcine skeletal muscle

Ladybird-like genes were recently identified in mammals. The first member characterized, Lbx1, is expressed in developing skeletal muscle and the nervous system. However, little is known about the porcine Lbx1 gene. In the present study, we cloned and characterized Lbx1 from porcine muscle. RT-PCR analyses showed that Lbx1 was highly expressed in porcine skeletal muscle tissues. And we provide the first evidence that Lbx1 has a certain regulated expression pattern during the postnatal period of the porcine skeletal muscle development. Lbx1 gene expressed at higher levels in biceps femoris muscles compared with masseter, semitendinosus and longissimus dorsi muscles in Meishan pigs. Phylogenetic tree was constructed by aligning the amino acid sequences of different species. Moreover, single nucleotide polymorphism (SNP) scanning in the Lbx1 genomic fragment identified two mutations, g.752A>G and g.−1559C>G. Association analysis in our experimental pig populations showed that the mutation of g.752A>G was significantly associated with loin muscle area (P < 0.05) and internal fat rate (P < 0.05). Our results suggest that the Lbx1 gene might be a candidate gene of carcass traits and provide useful information for further studies on its roles in porcine skeletal muscle.     

The chromosomal localization,expression pattern and polymorphism analysis of porcine <Emphasis Type="Italic">FSCN1</Emphasis> gene differently expressed from LongSAGE library

Fascin homologue 1 (FSCN1) has established roles in cell adhesion, motility, and cell–cell interactions. Our LongSAGE analysis suggested that FSCN1 was potentially differentially expressed in prenatal pig skeletal muscle. We have cloned the genomic DNA and mRNA sequence of FSCN1 gene and mapped it to SSC3p16-p17. The FSCN1 gene was differently expressed during prenatal skeletal muscle development and exhibited different expression pattern between Tongcheng and Landrace pigs. In Tongcheng pigs, FSCN1 expression was similar at 33 and 65 days post conception (dpc), and then sharply increased to a peak at 90 dpc. In Landrace pigs, however, expression increased between 33 and 65 dpc, peaked at 65 dpc, and was down-regulated thereafter. Significantly different expression levels between Tongcheng and Landrace were observed at 65 and 90 dpc. In postnatal pigs, it was strongly expressed only in the brain, but weakly in skeletal muscle and other tissues. We initially identified 32 SNPs through genomic DNA of FSCN1 gene. Association analysis suggested that the 6840^C/T mutation was significantly associated with the age at market weight (AGE) (p = 0.0004), average day gain from birth to market (ADG1) (p = 0.0002), and average day gain at testing period (ADG2) (p < 0.0001). Our study suggested that FSCN1 gene plays an in prenatal skeletal muscle development and was a candidate gene for meat production trait.     

Molecular characterization and expression patterns of serine/arginine-rich specific kinase 3 (<Emphasis Type="Italic">SPRK3</Emphasis>) in porcine skeletal muscle

SRPK3 is a protein kinase belonging to serine/arginine protein kinases (SRPK) family, which phosphorylates serine/arginine repeat-containing proteins, and is controlled by a muscle-specific enhancer directly regulated by MEF2. In this study, a full-length cDNA of the porcine SRPK3 gene encoding a 566 amino acid protein was isolated. It contains 14 exons over approximately 4.3 kb. The deduced amino acid sequence of porcine SRPK3 contains a bipartite kinase domain, and shows high similarities to their corresponding human and cattle homologues. Tissue distribution analysis indicated that porcine SRPK3 mRNAs are highly expressed in heart and skeletal muscle especially in uterus and parorchis, but at low level in brain, stomach, small intestine, and ovary. Expression pattern of SRPK3 was similar in Large White and Chinese Meishan breeds. Both the two breeds had the highest expression levels at fetal 65 days (P < 0.01), and decreased while the age increased until 60 days old, then increased at 120 days (P < 0.01) and decreased at 180 days (P < 0.05). However, at fetal 65 days, the mRNA abundance of SRPK3 in Large White was 12.5-fold higher than in Meishan pigs (P < 0.01), whereas at 180 days, the abundance in Meishan was 3.4-fold higher than in Large White pigs (P < 0.01). These results suggest that the SRPK3 gene might be an important gene of skeletal muscle development and also provides basic molecular information useful for further studies on its roles in porcine skeletal muscle.     

Characterization of the porcine differentially expressed <Emphasis Type="Italic">PDK4</Emphasis> gene and association with meat quality

To investigate the differential expression of genes in the skeletal muscle between Yorkshire and Chinese indigenous breed Meishan pigs, suppression subtractive hybridization was carried out and many genes were proved to be expressed significantly different in the two breeds. One gene highly expressed in Meishan but lowly expressed in Yorkshire specific library, shared strong homology with human pyruvate dehydrogenase kinase 4 (PDK4). Using semi-quantity and quantity PCR, We confirmed its differential expression between the two breeds. Temporal and spatial expression analysis indicated that porcine PDK4 gene is highly expressed in skeletal muscle and the highest in neonatal pigs. Complete cDNA cloning and sequence analysis revealed that porcine PDK4 gene contains an open reading frame of 1,221 bp. The deduced amino acid sequence showed conservation in evolution. A G/A mutation in intron 9 was identified and association analysis showed that it was significantly associated with intramuscular fat, muscle water content.     

Molecular characterization,expression pattern and association analysis of the porcine <Emphasis Type="Italic">BTG2</Emphasis> gene

B-cell translocation gene 2 (BTG2), a member of the B-cell translocation gene family with anti-proliferative properties, have been characterized to be involved in cell growth, differentiation and survival. In this study, we cloned the full length sequences of cDNA and genomic DNA of BTG2 gene from the porcine skeletal muscle. Spatial expression analysis showed that the porcine BTG2 gene is expressed predominantly in muscle. Temporal expression analysis in longissimus dorsi muscle demonstrated that the expression of BTG2 gene has the highest expression at 60 days old in Large White while with a peak expression at 120 days old in Meishan. Temporal analysis also revealed that the expression of BTG2 gene is generally higher in Large White than in Meishan at all the developmental stages tested (65 days of conception and 3, 35, 60, 120, and 180 days of postnatal). A single nucleotide polymorphism (G417C) in the intron of BTG2 gene was then detected by PCR-RFLP in Large White × Meishan F2 resource population and association analysis suggested that this polymorphic site had significant association (P < 0.05) with the buttock fat thickness, fat percentage, lean muscle percentage, ratio of lean to fat and carcass length.     

Cloning,chromosomal localization,SNP detection and association analysis of the porcine IRS-1 gene

Insulin receptor substrate-1(IRS-1) gene is one member of the Insulin receptor substrate (IRS) gene family, which plays an important role in mediating the growth of skeletal muscle and the molecular metabolism of type 2 diabetes. Here, we cloned a 3,573 bp fragment of the partial CDS sequence of porcine IRS-1 gene by in silicon cloning strategy and RT-PCR method. The porcine IRS-1 gene was assigned to SSC15q25 by using IMpRH. Sequencing of PCR products from Duroc and Tibetan pig breeds identified one SNP in exon 1 of porcine IRS-1 gene (C3257A polymorphisms). Association analysis of genotypes with the growth traits, anatomy traits, meat quality traits and physiological biochemical indexes traits showed that different genotypes at locus 3,257 of IRS-1 have significant differences in carcass straight length in pigs (P = 0.0102 < 0.05).     

Functional and association studies of the cholesteryl ester transfer protein (CETP) gene in a Wannan Black pig model

Some polymorphisms of the human CETP gene are causally and significantly associated with serum lipids levels; however, the information regarding this gene in pigs is sparse. To evaluate the effects of CETP on blood lipid traits and fat deposition in pig, porcine CETP tissue expression patterns were observed by quantitative real‐time polymerase chain reaction (qPCR) first. High expression was detected in liver, spleen, gluteus medius (GM) muscle and backfat. A de novo polymorphism (AF333037:g.795C>T) in the intron 1 region of porcine CETP was identified. This polymorphism was further genotyped by direct sequencing of the PCR products of 390 Wannan Black pigs, a Chinese native breed population. Association analyses at 45 and 300 days of age revealed highly significant associations between CETP genotypes and serum lipid traits. Furthermore, this polymorphism was proved to be associated with differences in liver CETP mRNA levels: pigs at 300 days of age with the TT genotype had higher levels than did those with other genotypes (P = 0.021). Additionally, analysis at 300 days of age showed that GM CETP mRNA expression correlated positively with serum lipids levels as well as with carcass backfat thickness and intramuscular fat content in GM. These results indicate that CETP is involved in serum, adipose and muscle lipid metabolism in pigs. The mechanisms underlying such relationships and their functional implications are worthy of further research.     

Imprinting analysis of porcine MAGEL2 gene in two fetal stages and association analysis with carcass traits

Imprinted genes play an essential role in the regulation of fetal growth, development and function of the placenta, however only a limited number of imprinted genes have been studied in swine. In this study, we cloned and characterized porcine MAGEL2 (melanoma antigen-like gene 2), and also identified its imprinting status during porcine fetal development. The complete open reading frame (ORF) encoding 1,193 amino acids was isolated and two single nucleotide polymorphisms (SNPs) (g.2592A>C and g.3277T>C) in the coding region were identified. The reciprocal Yorkshire × Meishan F₁ hybrid model and the RT-PCR/RFLP method were used to detect the imprinting status of porcine MAGEL2 gene at two developmental stages of day 30 and 65 of gestation. Imprinting analysis showed that porcine MAGEL2 was paternally expressed in day 65 fetal tissues, including heart, liver, spleen, lung, kidney, stomach, small intestine, skeletal muscle, brain and placenta. Interestingly, we observed an imprinting variance of MAGEL2 gene in 30 dpc fetuses produced by the cross of Yorkshire boar × Meishan sow, in which seven heterozygous fetuses were monoallelically expressed from the paternal allele but two were biallelically expressed from both the paternal and maternal alleles. Association analysis in a Yorkshire × Meishan F₂ resource population showed that the mutation of g.2592A>C was significantly associated with dressed carcass percentage (P < 0.05) and buttock fat thickness (P < 0.05). Our results suggest that MAGEL2, as a novel imprinted gene in pig, might be a candidate gene affecting carcass traits and could provide important information for the functional study of imprinted genes during porcine development.     

Application of iTRAQ to catalogue the skeletal muscle proteome in pigs and assessment of effects of gender and diet dephytinization

In this study iTRAQ was used to produce a highly confident catalogue of 542 proteins identified in porcine muscle (false positive<5%). To our knowledge this is the largest reported set of skeletal muscle proteins in livestock. Comparison with human muscle proteome demonstrated a low level of false positives with 83% of the proteins common to both proteomes. In addition, for the first time we assess variations in the muscle proteome caused by sexually dimorphic gene expression and diet dephytinization. Preliminary analysis identified 19 skeletal muscle proteins differentially expressed between male and female pigs (≥1.2‐fold, p<0.05), but only one of them, GDP‐dissociation inhibitor 1, was significant (p<0.05) after false discovery rate correction. Diet dephytinization affected expression of 20 proteins (p<0.05). This study would contribute to an evaluation of the suitability of the pig as a model to study human gender‐related differences in gene expression. Transgenic pigs used in this study might also serve as a useful model to understand changes in human physiology resulting from diet dephytinization.     

The microRNA expression profile in porcine skeletal muscle is changed by constant heat stress

Heat stress has profound effects on animal performance and muscle function, and microRNAs (miRNAs) play a critical role in muscle development and stress responses. To characterize the changes in miRNAs in skeletal muscle responding to heat stress, the miRNA expression profiles of longissimus dorsi muscles of pigs raised under constant heat stress (30 °C; n = 8) or control temperature (22 °C; n = 8) for 21 days were analyzed by Illumina deep sequencing. A total of 58 differentially expressed miRNAs were identified with 30 down‐regulated and 28 up‐regulated, and 63 differentially expressed target genes were predicted by miRNA–mRNA joint analysis. GO and KEGG analyses showed that the genes regulated by differentially expressed miRNAs were enriched in glucose metabolism, cytoskeletal structure and function and stress response. Real‐time PCR showed that the mRNA levels of PDK4, HSP90 and DES were significantly increased, whereas those of SCD and LDHA significantly decreased by heat exposure. The protein levels of CALM1, DES and HIF1α were also significantly increased by constant heat. These results demonstrated that the change in miRNA expression in porcine longissimus dorsi muscle underlies the changes in muscle structure and metabolism in porcine skeletal muscle affected by constant heat stress.     

A genome-wide association study revealed five SNPs affecting 8-month weight in sheep

Lamb weight at 8 months of age is an important trait in the sheep industry in terms of the onset of puberty around this age; however, knowledge of its effective genetic factors is limited. Therefore, a GWAS using the 50K SNP-Chip was performed on 96 Baluchi sheep to identify the genomic regions associated with 8-month weight. The results of the present study revealed five SNPs on chromosomes 4, 14 and 16 at 5% chromosome-wide significance level, jointly accounting for 0.95% of total genetic variance. Four genes – MTPN, HYDIN, LRGUK and ZFP90 – were found in 50 kb intervals around the significant SNPs, of which MTPN is involved in regulation of skeletal muscle growth. Our results may provide a new vision to identify the genomic regions affecting growth traits in sheep.     

Dietary arginine supplementation enhances antioxidative capacity and improves meat quality of finishing pigs

The present study was conducted to test the hypothesis that dietary arginine supplementation may improve meat quality of finishing pigs. Beginning at ~60 kg body weight, pigs were fed a corn- and soybean meal-based diet supplemented with 0, 0.5 or 1% l-arginine until they reached a body weight of ~110 kg. On the last day of the experiment, pigs were food-deprived for 16 h before blood samples were obtained for analysis of amino acids, insulin, and other metabolites. Immediately thereafter, pigs were slaughtered for determination of carcass composition, muscle biochemical parameters, and meat quality. The result showed that arginine did not affect pig growth performance or carcass traits. However, 1% arginine decreased drip loss of pork muscle at 48 h postmortem, while increasing intramuscular fat content (P < 0.05). Supplementing 0.5 or 1% arginine to the diet increased arginine concentration and decreased cortisol level in serum, while enhancing antioxidative capacity and glutathione peroxidase activity in serum (P < 0.05). Additionally, 1% arginine increased antioxidative capacity in skeletal muscle (P < 0.05). Furthermore, 0.5 or 1% arginine decreased the cortisol receptor mRNA level in muscle (P < 0.05). Collectively, these results indicate that supplemental arginine improved meat quality and attenuated oxidative stress of finishing pigs.