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.     

Molecular characterization of differentially expressed TXNIP gene and its association with porcine carcass traits

Thioredoxin interacting protein (TXNIP), which plays a regulatory role in lipid metabolism and immune regulation, is down-regulated expressed in F₁ hybrids Landrace?×?Yorkshire skeletal muscle. Here we described the molecular characterization of porcine TXNIP gene. The full-length cDNA contains a coding sequence of 1,176?bp nucleotides with untranslated regions of 263?bp at 5′-end and 441?bp at 3′-end, respectively. The predicted molecular mass and isoelectric point of porcine TXNIP is 43.81?kDa and 7.385, respectively. The deduced 391 amino acids exhibit high identity with other mammalian TXNIP. The TXNIP gene contains eight coding exons and seven non coding introns, spans approximately 3,348?bp. The expression of porcine TXNIP mRNA is almost absent in Landrace?×?Yorkshire and lower level in 6-month-old pigs during skeletal muscle development. Other stages and breeds were high level expressed. Statistical analysis showed the TXNIP gene polymorphism (c.575-4T>C) was different between F₁ hybrids and their parents, was highly associated with dressing percentage (DP) and thorax–waist fat thickness (TFT) in the Yorkshire?×?Meishan F₂ population. The possible role of TXNIP was discussed.     

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.     

Assessment of genomic imprinting of <Emphasis Type="Italic">PPP1R9A</Emphasis>, <Emphasis Type="Italic">NAP1L5</Emphasis> and <Emphasis Type="Italic">PEG3</Emphasis> in pigs

Imprinted genes play significant roles in the regulation of fetal growth and development, function of the placenta, and maternal nurturing behaviour in mammals. At present, few imprinted genes have been reported in pigs compared to human and mouse. In order to increase understanding of imprinted genes in swine, a polymorphism-based approach was used to assess the imprinting status of three porcine genes in 12 tissue types, obtained from F1 pigs of reciprocal crosses between Rongchang and Landrace pure breeds. In contrast to human and mouse homologues, porcine PPP1R9A was not imprinted, and was found to be expressed in all tissues examined. The expression of porcine NAP1L5 was detected in pituitary, liver, spleen, lung, kiduey, stomach, small intestine, skeletal muscle, fat, ovary, and uterus, but undetectable in heart. Furthermore, porcine NAP1L5 was paternally expressed in the tissues where it’s expression was observed. For PEG3, pigs expressed the paternal allele in skeletal muscle, liver, spleen, kidney, and uterus, but biallele in heart, lung, fat, stomach, small intestine, and ovary. Our data indicate that tissue distribution of the three gene differs among mammals, and the imprinting of NAP1L5 and PEG3 is well conserved.     

Expression pattern and polymorphism of three microsatellite markers in the porcine CA3 gene

Carbonic anhydrase III (CA3) is an abundant muscle protein characteristic of adult type-1, slow-twitch, muscle fibres. In order to further understand the functions of the porcine CA3 protein in muscle, the temporal and spatial distributions of its gene product were analysed and the association between the presence of specific polymorphisms and carcass traits in the pig was also examined. Real-time PCR revealed that the CA3 mRNA expression showed no differences with age in skeletal muscles from Yorkshire pigs at postnatal day-1, month-2, and month-4. We provide the first evidence that CA3 is differentially expressed in the skeletal muscle of Yorkshire and Meishan pig breeds. In addition, the whole pig genomic DNA sequence of CA3 was investigated and shown to contain seven exons and six introns. Comparative sequencing of the gene from three pig breeds revealed the existence of microsatellite SJ160 in intron 5 and microsatellite SJ158 and a novel microsatellite marker that includes a tandem repeat of (TC)_n in intron 4. We also determined the allele number and frequencies of the three loci in seven pig breeds and found that they are low polymorphic microsatellite markers. Statistical analysis showed that the CA3 microsatellite polymorphism was associated with dressing percentage, internal fat rate, carcass length, rib number and backfat thickness in the pig.     

Porcine skeletal muscle differentially expressed gene ATP5B: molecular characterization, expression patterns, and association analysis with meat quality traits

The 2-DE/MS-based proteomics approach was used to investigate the differences of porcine skeletal muscle, and ATP5B was identified as one differential expression protein. In the present study, ATP5B gene was further cloned by RT-PCR, the sequence was analyzed using the bioinformatics method, and the mRNA expression was detected by qRT-PCR. Sequence analysis showed that the porcine ATP5B gene contains an ORF encoding 528-amino-acid residues with 49 and 166 nucleotides in the 5′ and 3′ UTRs, respectively. The mRNA of ATP5B was widely expressed in all 14 tissues tested, but especially highly expressed in parorchis and fat. The expression pattern of ATP5B was similar in Large White and Meishan breeds, showing that the expression was upregulated by 3 days after birth and downregulated during postnatal development of skeletal muscle. Comparing the two breeds, the mRNA abundance of ATP5B in Large White was more highly expressed than in Meishan at all developmental stages (P < 0.05). Moreover, a synonymous mutation, G75A in exon 8, was identified and association analysis with the traits of meat quality showed that it was significantly associated with the RLF, FMP, IFR, IMF, and IMW (P < 0.05). These results suggested that ATP5B probably plays a key role in porcine skeletal muscle development and may provide further insight into the molecular mechanisms responsible for breed-specific differences in meat quality.     

Molecular characterization, expression patterns, and polymorphism of a differentially expressed porcine gene (PYGM) isolated by suppression subtractive hybridization and two-dimensional gel electrophoresis analysis

Suppression subtractive hybridization was performed to detect the differences in gene expression of porcine longissimus dorsi muscles between Large White and Chinese Meishan pigs. An upregulated gene in Large White that shared high homology with human muscle glycogen phosphorylase (PYGM) was identified. The porcine PYGM gene contains an open reading frame encoding 842 amino acid residues with 26 and 283 nucleotides in the 5' and 3' untranslated regions, respectively. Tissue distribution analysis indicated that porcine PYGM mRNAs are highly expressed in all tissues. Expression pattern of PYGM was similar in the two breeds. Both breeds had the highest expression levels when 120 days old (p<0.01), and PYGM was upregulated during skeletal muscle development. A similar expression pattern of PYGM in protein level was also observed by differential proteome analysis of skeletal muscle development using two-dimensional gel electrophoresis and mass spectroscopy. The mRNA abundance of PYGM in Large White was higher than Meishan at all four stages (p<0.05). Moreover, a G/T mutation in exon 8 was identified and association analysis with meat quality traits showed that it was significantly associated with lean meat percentage (p<0.05). Our data may provide further insight into the molecular mechanisms responsible for breed-specific differences in porcine growth and meat quality.     

Molecular characterization,expression patterns and subcellular localization of Myotrophin (MTPN) gene in porcine skeletal muscle

Myotrophin (MTPN) is an effective growth factor in promoting skeletal muscle growth in vitro and vivo and has been purified from porcine skeletal muscle. However, in pigs, the information on MTPN gene is very limited. In this study, we cloned cDNA sequences and analyzed the genomic structure of porcine MTPN gene. The deduced amino acid sequence of porcine MTPN contains two the ankyrin repeat domains. RT-PCR analysis revealed that porcine MTPN gene was widely expressed in many tissues, a high expression level was observed in the spleen, liver and uterus, and transient transfection indicated that porcine MTPN proteins was located in cytoplasms within Pig Kidney Epithelial cells (PK15). Quantitative real-time PCR (qRT-PCR) analyses showed that MTPN expression peaked at embryonic 65 day post conception (dpc). During postnatal muscle development, MTPN expression was down-regulated from the 3 day to the 180 day in Yorkshire pigs. This result suggests that the MTPN gene may be important gene for skeletal muscle growth and provides useful information for further studies on its roles in porcine skeletal muscle.     

Molecular cloning,polymorphism and association analyses of a novel differentially expressed porcine mRNA

The mRNA differential display technique was performed to investigate the differences of gene expression in the longissimus muscle tissues from Meishan and Large White pigs. One novel mRNA that was differentially expressed was identified through semi-quantitative RT-PCR and the full-length cDNA sequence was then obtained using the rapid amplification of cDNA ends (RACE) method. The nucleotide sequence of the mRNA is not homologous to any of the known porcine genes. Sequence prediction analysis revealed that this mRNA is no-coding mRNA. Polymorphism analyses revealed that there was a C-T mutation on the position of 505 bp and PCR-HhaI-RFLP analyses revealed that Chinese indigenous pig breeds and exotic pig breeds displayed obvious genotype and allele frequency differences at this locus. Association analyses revealed that this polymorphic locus was significantly associated with the drip loss rate, water holding capacity, dressing percentage, rib numbers, lean meat percentage, estimated lean meat percentage, loin eye width and loin eye area (P < 0.05).     

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.     

Investigation of <Emphasis Type="Italic">Lpin1</Emphasis> as a candidate gene for fat deposition in pigs

Lpin1 deficiency prevents normal adipose tissue development and remarkably reduces adipose tissue mass, while overexpression of the Lpin1 gene in either skeletal muscle or adipose tissue promotes adiposity in mice. However, little is known about the porcine Lpin1 gene. In the present study, a 5,559-bp cDNA sequence of the porcine Lpin1 gene was obtained by RT-PCR and 3′RACE. The sequence consisted of a 111-bp 5′UTR, a 2,685-bp open reading frame encoding a protein of 894 amino acids and a 2,763-bp 3′UTR. Semi-quantitative RT-PCR analysis revealed that Lpin1 had a high level of expression in the liver, spleen, skeletal muscle and fat, a low level of expression in the heart, lung and kidney. The porcine Lpin1 gene was assigned to 3q21-27 by using the somatic cell hybrid panel (SCHP) and the radiation hybrid (IMpRH) panel. One C93T single nucleotide polymorphism (SNP) was identified and genotyped using the TaqI PCR-RFLP method. Association analysis between the genotypes and fat deposition traits suggested that different genotypes of the Lpin1 gene were associated with percentage of leaf fat and intramuscular fat.