Differential expression and effect of the porcine ANGPTL4 gene on intramuscular fat.

In a previous study, we investigated differences in gene expression in backfat between Meishan and Large White pigs and their F1 hybrids, Large White x Meishan, and Meishan x Large White pigs. One potential differentially expressed sequence tag from the mRNA differential display was a homolog of the human angiopoietin-like 4 (ANGPTL4) gene, which encodes a protein that is secreted by both liver and white adipose tissues and can inhibit lipoprotein lipase activity and stimulate white adipose tissue lipolysis. Here, ANGPTL4 mRNA was found to be upregulated in the backfat of Large White compared with that in the Meishan pigs and the F1 hybrids, Meishan x Large White and Large White x Meishan, whereas expression was lowest both in the longissimus dorsi and the heart, as shown by the tissue distribution profile. Only one mutation, a G/A transition located in the third intron, was found. The ANGPTL4 G/A polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) showed a significant effect on intramuscular fat (IMF), water moisture of the longissimus dorsi, meat marbling of the longissimus dorsi, and pH of the longissimus dorsi (P < 0.05). This site seemed to be significantly (P < 0.05) additive in its actions on IMF, water moisture, and pH, whereas it showed significant dominance in its action on meat marbling (P < 0.05). This locus can be potentially considered as a marker for IMF improvement.

Expression patterns and promoter activity analysis of UGP2 in pigs.

The function of the UDP-glucose pyrophosphorylase 2 gene (UGP2) in pig is not clear. In the present study, we used RNA isolated from Large White pigs and Chinese indigenous MeiShan pigs to examine the temporal coordination of changes in gene expression within muscle tissues. We cloned both the complete genomic DNA sequence and 2077-bp 5ꞌ-flanking sequence of porcine UGP2, to determine the genomic sequence. Real-time RT-PCR revealed that UGP2 was highly expressed in liver and skeletal muscle of MeiShan pigs. Among different types of muscle fibers, the UGP2 had the highest expression in both soleus muscle and longissimus dorsi in Large White pigs. In the progression of muscle fibers at different growth stages, UGP2 plays a role in the early days after birth in Large White pigs, while in MeiShan pigs it is important later. Furthermore, the 5ꞌ-flanking sequence we cloned exhibited the promoter activity of UGP2, and the sequence 588 bp upstream from the transcriptional site had the greatest activity.

Real-time reverse transcription-PCR expression profiling of porcine troponin I family in three different types of muscles during development.

In this study, the expression profiling of three troponin I isoforms (TNNI1, TNNI2 and TNNI3) was investigated in two pig breeds differing in muscularity (Yorkshire and Meishan) at six stages (fetal 60 days and postnatal 3, 35, 60, 120, and 180 days) and three types of muscles (longissimus dorsi muscle, LD; semitendinosus, ST; cardiac muscle, CM) using relative real-time quantitative PCR. Significant differences of troponin I expression in three muscles were found between Yorkshire and Meishan breeds at some stages. The expression peak of TNNI1 and TNNI2 in LD and ST was at postnatal 35 or 60 days in Yorkshire and at postnatal 120 or 180 days in Meishan pigs, while it occurred in CM at postnatal 3 days in two pig breeds. The relative expression values of TNNI1 and TNNI2 were significantly higher in LD than ST at most of stages after birth. The expression ratio of TNNI2 versus TNNI1 favoured TNNI2 expression in ST and LD, but on the contrary in CM. The expression peak of TNNI3 occurred at postnatal 60 and 120 days in Yorkshire and Meishan pigs, respectively. TNNI1 and TNNI3 were co-expressed in CM during the fetal and earlier stages after birth.

Molecular characterization, expression pattern and association analysis of the porcine BTG2 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.

Association of 3 polymorphisms in porcine troponin I genes (TNNI1 and TNNI2) with meat quality traits.

The contractile protein troponin I (TnI), a constituent protein of the troponin complex located on the thin filaments of striated muscle, is involved in inhibition of calcium-induced myosin ATPase activity (and thus contraction). TnI-slow (slow-twitch skeletal muscle isoform, named TNNI1) and TnI-fast (fast-twitch skeletal muscle isoform, named TNNI2) are muscle-fiber-type-specific proteins, and expression of their genes may affect the composition of muscle fiber, thereby influencing the meat quality traits. Thus, the TnI genes are potential candidate genes for traits related to meat quality in animals. Association of 2 SNPs (EU743939:g.5174T>C in intron 4, and EU743939:g.8350C>A in intron 7) of the TNNI1 gene and a SNP (EU696779:g.1167C>T in intron 3) of the TNNI2 gene with 11 meat quality traits were studied on 334 Large White x Meishan F(2) pigs. In the TNNI1 gene, g.5174T>C and g.8350C>A were found to be significantly associated with intramuscular fat content and meat color value of biceps femoris. The g.5174T>C also showed significant effects on meat color value and marbling score of longissimus dorsi, as well as pH of longissimus dorsi and semispinalis capitis. The g.1167C>T polymorphism in the TNNI2 gene affected significantly the pH of longissimus dorsi, meat color value of longissimus dorsi and semispinalis capitis, marbling score of longissimus dorsi, and intramuscular fat.

Identification of three novel SNPs and association with carcass traits in porcine TNNI1 and TNNI2.

In this study, two novel SNPs (EU743939:g.5174T>C in intron 4 and EU743939:g.8350C>A in intron 7) in TNNI1 and one SNP (EU696779:g.1167C>T in intron 3) in TNNI2 were identified by PCR-RFLP (PCR restriction fragment length polymorphism) using XbaI, MspI and SmaI restriction enzyme, respectively. The allele frequencies of three novel SNPs were determined in the genetically diverse pig breeds including ten Chinese indigenous pigs and three Western commercial pig breeds. Association analysis of the SNPs with the carcass traits were conducted in a Large White × Meishan F(2) pig population. The linkage of two SNPs (g.5174T>C and g.8350C>A) in TNNI1 gene had significant effect on fat percentage. Besides these, the g.5174T>C polymorphism was also significantly associated with skin percentage (P < 0.05), shoulder fat thickness (P < 0.05) and backfat thickness between sixth and seventh ribs (P < 0.05). The significant effects of g.1167C>T polymorphism in TNNI2 gene on fat percentage (P < 0.01), lean meat percentage (P < 0.05), lion eye area (P < 0.05), thorax-waist backfat thickness (P < 0.01) and average backfat thickness (P < 0.05) were also found.

Molecular characterization and association with carcass traits of the porcine SLC39A7 gene.

In this study, the molecular characterization and potential association of SLC39A7 gene with carcass traits were investigated in pigs. The sequence of SLC39A7 cDNA was obtained by in silico cloning and RT-polymerase chain reaction (PCR). Two transcripts, variant 1 (2398 bp) and variant 2 (2088 bp), of the SLC39A7 gene were identified. Expression analysis of SLC39A7 in 10 different tissues by RT-PCR showed that variant 1 was ubiquitously expressed in all tissues analysed, but variant 2 was not found in fat tissue. The cDNA regions of variant 1 and 2 were organized in seven and eight exons respectively. A c.205G>A substitution in exon 3, which changes a codon for glycine into a codon for arginine, (p.Gly69Arg) and a c.1138-216T>C substitution in intron 6 were detected by PCR-HpaII-restriction fragment length polymorphisms (RFLP) and PCR-cofI-RFLP respectively. Significant differences were found in the allele frequencies of c.205G>A among six Chinese indigenous pig breeds and two commercial pig breeds. Linkage analysis showed that the c.205G>A polymorphism within the SLC39A7 gene was closely linked to the marker Sw1856 on pig chromosome 7 in a Large White x Meishan F(2) resource population. The QTL and association studies between polymorphisms of the SLC39A7 gene and carcass traits were carried out. Significant associations of the SLC39A7 polymorphisms with backfat thickness at thorax-waist (p < 0.05), average backfat thickness (p < 0.05) and leaf fat weight (p < 0.01) were found. Additional F-drop test or marker assisted association analyses also supported the association of the mutation in SLC39A7 with the above traits. Together, the present study provided the useful information for the characterization of SLC39A7 gene and potential association with carcass traits in pigs.

Differential proteome analysis of porcine skeletal muscles between Meishan and Large White.

Western and indigenous Chinese pig breeds show obvious differences in muscle growth and meat quality; however, the underlying molecular mechanism remains unclear. In this study, proteome analysis of LM between purebred Meishan and Large White pigs was performed by 2-dimensional gel electrophoresis and mass spectrometry. A total of 25 protein spots were differentially expressed in the 2 breeds. The 14 identified proteins could be divided into 4 groups: energy metabolism, defense and stress, myofibrillar filaments, and other unclassified proteins. Quantitative real-time PCR was used to analyze the partly differentially expressed proteins in mRNA level, which revealed a positive correlation between the content of the proteins and their mRNA levels. We also analyzed the mRNA levels of myosin heavy chain isoforms using quantitative real-time PCR. The results indicated that IIa and IIx fibers were elevated in Meishan pigs, whereas the IIb fiber was more highly expressed in Large White pigs. To the best of our knowledge, this was the first proteomics-based investigation of total skeletal muscle protein in different pig breeds, and these results may provide valuable information for understanding the molecular mechanism responsible for breed-specific differences in growth performance and meat quality.

Genetic polymorphisms and preliminary association analysis with production traits of the porcine SLC27A4 gene.

Solute carrier family 27 (fatty acid transporter), member 4 (SLC27A4) is a fatty acyl-CoA synthetase producing very long chain fatty acid-CoA for lipid metabolic pathways, suggesting that the SLC27A4 gene is a potential candidate gene for traits related to fat deposition in animals. This study was conducted to sequence the genomic region from exon 6 to 12 of porcine SLC27A4 and detect polymorphisms by comparative sequencing. In silico mapping assigned SLC27A4 gene between gene COQ4 (coenzyme Q4 homolog) and URM1 (ubiquitin related modifier 1 homolog) on pig chromosome 1q24-q2.12 where significant QTL affecting backfat depth had previously been identified. Thirty six putative sites of variation were detected, of which 31 polymorphisms including 28 SNPs and 3 indels were located in the intronic region, and 5 in the exonic regions. The g.1777G>A (EU703769) in intron 8 was confirmed by PCR-RFLP using HpaII restriction enzyme and further genotyped in four Chinese native pig breeds (Meishan, Erhualian, Tongcheng and Qingping) and three western meat-type pig breeds (Duroc, Large White and Landrace). Allele G was exclusively present in Tongcheng and Qingping pigs and predominant in the other pig populations analyzed. Significant differences of backfat at rump, body weight at birth and average daily gain on weaning between the AG and GG genotype were observed in Landrace pig population (P < 0.05).

Molecular characterization and SNPs analysis of the porcine Deleted in AZoospermia Like (pDAZL) gene.

The Deleted in AZoospermia Like (DAZL) gene is expressed in prenatal and postnatal germ cells. In this study, we cloned and characterized the porcine Deleted in AZoospermia Like (pDAZL) gene. We found the full-length coding sequence of the pDAZL encoded a protein of 295 amino acids with a RNA recognition motif (amino acids 41-111) and a DAZ repeat (amino acids 167-120). The deduced protein sequence of pDAZL is 92.5% and 91.5% similar to those of human and bovine, respectively. PCR-MspI-RFLP and PCR-TaqI-RFLP were established to detect an A/G mutation in intron 7 and a C/A mutation in intron 9, respectively. Associations of two SNPs with litter size traits were assessed in Large White (n=275) and DIV (n=128) pig populations, and the statistical analysis demonstrated that CC produced 0.716 more (P<0.05) piglets born alive than CD genotypes in Large White pigs at TaqI locus (C/A mutation in intron 9), and the dominance effect was 0.304 pig per litter (P<0.05). This result suggests that the pDAZL gene might be a good candidate gene of litter size trait and provides some marker information for marker-assisted selection (MAS).

Identification of polymorphism and association analysis with reproductive traits in the porcine RNF4 gene.

The ring finger protein 4 gene (RNF4), which might play a role in fetal germ cell development as well as in oocyte and granulosa cell maturation, was one of the potential candidate genes for reproductive traits. In the present work, we isolated the complete coding sequence of porcine RNF4 gene, identified a single nucleotide polymorphism (SNP: T/C) in intron5, and developed a PCR-SacII-RFLP genotyping assay. Association of this SNP with reproductive traits was assessed in three populations with diverse genetic backgrounds. One was Chinese Qingping sows. Another was consisted of crossbred sows derived from Landrace, Large White, Chinese Tongcheng and/or Chinese Meishan (Line DIV). The third is Large White x Meishan (LW x M) F(2) slaughtered population. Statistical analysis demonstrated that, in the first parity, the difference between RNF4 genotypes and reproductive traits of both Qingping and Line DIV sows was not significant. In the second and subsequent litters, CC animals in Qingping population had more piglets born (+1.74 piglets) and piglets born alive (+2.02 piglets) than sows with the TT genotype (P<0.05). Line DIV sows inheriting the CC genotype had additional 0.69 piglets born compared to the TC animals (P<0.05) in second and subsequent litters. No significant difference was observed between genotypes and reproductive tracts components in F(2) animals. In addition, we found RNF4 gene has a significant additive effect on both piglet born and piglet born alive in Qingping animals (P<0.05). Results here suggested that the RNF4 SNP was significantly associated with litter size in two populations and could be useful in selection for increasing litter size in pigs. Further studies were needed to confirm these preliminary researches.

Molecular cloning and polymorphism of the porcine H2AFZ gene.

H2A histone family, member Z (H2A.Z) is required for early mammalian development. In the present study, the 932 bp of full-length cDNA encoding a 128 amino-acid protein and the sequences of intron 2 to 4 of the porcine H2A histone family, member Z (pH2AFZ) gene were obtained. By comparative sequencing of pH2AFZ gene in Large White and Meishan pigs, a 4 bp deletion/insertion in intron 2 was detected and a PCR-Bsu15I-RFLP was established to detect this variation. In DIV (4th Dam line of Chinese lean-type new lines) pigs, the first-parity females with AA genotype had fewer piglets born alive (-2.64 and -1.83 piglets per litter) than those with AB (P < 0.01) and BB (P < 0.05) genotype. The additive allelic and dominance effect were estimated to be 0.92 (P < 0.05) and -0.87 piglets per litter (P < 0.01) for number of piglets born alive, respectively. This result suggests that the pH2AFZ gene might be a good candidate gene of litter-size trait and provides some marker information for marker-assisted selection.

Imprinted status of pleomorphic adenoma gene-like I and paternal expression gene 10 genes in pigs.

Genomic imprinting is theorized to exist in all placental mammals and some marsupials. Imprinted genes play important roles in the regulation of fetal growth, development, and postnatal behavior, but the study of imprinted genes has been limited in livestock. In this study, the polymorphism-based approach was used to detect the expression patterns of the porcine pleomorphic adenoma gene-like I (PLAGL1) and paternal expression gene 10 (PEG10) genes. Single nucleotide polymorphisms in the exons were detected between the Meishan and Large White breeds in the PLAGL1 and PEG10 genes. The polymorphisms were used to determine the monoallelic or biallelic expression with reverse transcription-PCR-RFLP in 44 tissues from 4 heterozygous pigs (based on SNP). Imprinting analysis indicated that the PLAGL1 and PEG10 genes were both paternally expressed in all tissues tested (heart, liver, spleen, lung, kidney, stomach, small intestine, skeletal muscle, fat, uterus, and ovary). Our study showed that the method of identifying polymorphic transcripts with reverse transcription-PCR-RFLP may be beneficial for detecting the imprinting status of some candidate imprinted genes.

Association of the polymorphism in GYS1 and ACOX1 genes with meat quality traits in pigs.

Phenotypic information about several pig meat quality traits on 334 Large White × Meishan F2 pigs was collected. Effects of the association of the FokI variants in the seventh intron of the skeletal muscle glycogen synthase (GYS1) gene and the PstI variants in the ninth intron of the palmitoyl acyl-CoA oxidase 1 (ACOX1) gene on the meat quality traits were examined on all pigs. The FokI variants of the GYS1 gene showed significant effects on pH of m. semipinalis capitis (P < 0.05). Linkage analysis indicated that the peak of the quantitative trait loci (QTL) curve was located around this marker for pH, but it did not reach significance (P > 0.05). The results may be due to several reasons such as linkage disequilibrium to the causal mutations, the limited number of animals or balance of another QTL or marker with negative effects. Significant effects of PstI variants of ACOX1 gene were also found on meat colour value and meat marbling score of both m. longissimus dorsi and m. biceps femoris (P < 0.05). Dominant effects for the affected traits at those two loci were significant except for meat marbling score of m. biceps femoris (P < 0.05). The results of this study give us some evidence for the potential of those dominant markers used in the marker-assisted selection of crossbreeding of the Large White pig sire lines and Meishan-derived synthetic dam lines.

Identification of a differential gene HUMMLC2B between F1 hybrids Landrace x Yorkshire and their female parents Yorkshire.

In order to investigate heterosis on a molecular basis, suppression subtractive hybridization was used to analyze the differences in gene expression between porcine F1 hybrids Landrace x Yorkshire and their female parents Yorkshire. From two specific subtractive cDNA libraries, the clones screened out by reverse Northern high-density blots screening were chosen to clone full-length cDNA by RACE. An expression-upregulated gene for Yorkshire skeletal muscle, designated as HUMMLC2B, was identified. Porcine HUMMLC2B contains an open reading frame (ORF) encoding 169 amino acids residues with 59 and 115 nucleotides in the 5' and 3' untranslated regions (UTRs), respectively. In the porcine genome, it contains seven exons separated by six introns. High allelic variations and four SINEs were detected in it. Comparison of derived amino acid sequence of HUMMLC2B with database sequences revealed highly conserved 12 amino acid residues in a putative calcium-binding region. RT-PCR analysis showed a tissue-specific pattern of expression in skeletal muscle and a similar level of expression during skeletal muscle development. The possible role of HUMMLC2B and its relation to porcine heterosis are discussed.

Polymorphism, linkage mapping and expression pattern of the porcine skeletal muscle glycogen synthase (GYS1) gene.

The glycogen synthase gene (GYS1), which encodes the rate-limiting enzyme for glycogen synthesis of skeletal muscle, is a promising candidate gene for traits related to skeletal muscle in pigs. In this study, a G/A single nucleotide polymorphism in GYS1 intron 7 detected as a FokI PCR-restriction fragment length polymorphism (PCR-RFLP) showed allele frequency differences among five Chinese indigenous pig breeds and three western commercial pig breeds. Linkage analysis assigned the gene GYS1 to marker interval SW1302-SW1473 on SSC6 in a three-generation Meishan X Large White reference family. The results of association analysis and interval mapping suggested that the FokI PCR-RFLP polymorphism might be linked with the quantitative trait loci affecting carcass traits detected on SSC6 in the F2 intercross pedigree. The reverse transcriptase-polymerase chain reaction revealed that the porcine GYS1 gene was expressed in spleen, lung, liver, kidney, small intestine, skeletal muscle, heart and stomach, with the highest expression in skeletal muscle.

cDNA cloning, genomic structure and polymorphism of the porcine FHL3 gene.

LIM domain proteins are important regulators of the growth, determination and differentiation of cells. Four-and-a-half LIM-only protein 3 (FHL3) is a type of LIM-only protein that contains four tandemly repeated LIM motifs with an N-terminal single zinc finger (half LIM motif). In this study, we have determined the complete coding sequence of pig FHL3 which encodes a 280 amino acid protein. The coding region of the pig FHL3 gene is organized in five exons and spans an approximately 2.1-kb genomic region. Comparative sequencing of six pig breeds revealed three single nucleotide polymorphisms (SNPs) within exon 2 of which an A-->G substitution at position 313 changes a codon for arginine into a codon for glycine. The substitution was situated within a PstI recognition site and developed as a PCR-RFLP marker for further use in population variation investigations and association analysis. The A/G polymorphism was segregating only in Landrace pigs. Association studies of the FHL3 polymorphism with carcass traits provided preliminary evidence that the PstI PCR-restriction fragment length polymorphism (RFLP) genotype may be associated with variation in several carcass traits of interest for pig breeding. Further investigations in more Landrace pigs are needed to confirm this.

Differential expression of caveolin-1 in lipopolysaccharide-activated murine macrophages.

Five reciprocal cycles of subtractive hybridization using cDNA generated from fibroblasts with normal lipopolysaccharide (LPS) responsiveness (lps(n)) and from hyporesponsive (lps(d)) fibroblasts have led to the finding that caveolin-1 is expressed at markedly higher levels of mRNA in lps(d) than in lps(n) fibroblasts. Caveolin-1 message can also be readily detected via reverse transcription-PCR in the RAW264.7 and J774.1 macrophage-like cell lines as well as in primary thioglycolate (TG)-elicited mouse peritoneal macrophages. In RAW264.7 cells, both caveolin-1 mRNA and protein levels are down-regulated by LPS. In TG-elicited C3HeB/FeJ peritoneal macrophages, in contrast, expression of both caveolin-1 protein and mRNA is up-regulated in vitro in response to LPS stimulation. The up-regulation of caveolin-1 protein expression in C3HeB/FeJ peritoneal macrophages can be demonstrated at concentrations as low as 1.0 pg of LPS/ml. However, LPS concentrations approximately 4 orders of magnitude higher (10(4) pg/ml) were required to stimulate the LPS-hyporesponsive C3H/HeJ mice peritoneal macrophages such that significant caveolin-1 protein up-regulation was detected. Caveolin-1, a principal component of plasmalemmal caveolae, has been reported as a potentially important regulator for signal transduction during cellular stimulation. The results described in this report suggest that caveolin-1 expression may be associated with LPS signaling/internalization.

Two functionally independent pathways for lipopolysaccharide-dependent activation of mouse peritoneal macrophages.

We have investigated the effects of human LPS-binding protein (LBP) and human bactericidal/permeability-increasing protein (BPI) on LPS-dependent activation of mouse thioglycolate-elicited peritoneal macrophages in vitro, in comparison with human PBMCs. Confirming earlier published studies, BPI inhibited, and LBP enhanced, the ability of LPS to stimulate PBMC production of the cytokines TNF-alpha and IL-6. In marked contrast to these results, under identical conditions of in vitro culture, both LBP and BPI suppressed, in a dose-dependent manner, the ability of LPS to stimulate cytokine production in mouse macrophages. Further, while human BPI also suppressed LPS-dependent NO secretion in mouse macrophages, human LBP had no inhibitory effect on NO secretion under conditions that inhibited TNF-alpha secretion. These data provide the first direct evidence that mouse macrophages may utilize two independent pathways in response to LPS, thus leading to different phenotypic responses.