Transcription factor C/EBPß promotes the transcription of the porcine GPR120 gene.

G protein-coupled receptor 120 (GPR120), an adipogenic receptor critical for the differentiation and maturation of adipocytes, plays an important role in controlling obesity in both humans and rodents and, thus, is an attractive target of obesity treatment studies. However, the mechanisms that regulate the expression of porcine GPR120 remain unclear. In this study, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) techniques were used to analyze and identify the binding of C/EBPß (transcription factor CCAAT/enhancer binding protein beta) to the GPR120 promoter. C/EBPß overexpression and RNA interference studies showed that C/EBPß regulated GPR120 promoter activity and endogenous GPR120 expression. The binding site of C/EBPß in the GPR120 promoter region from -101 to -87 was identified by promoter deletion analysis and site-directed mutagenesis. Overexpression of C/EBPß increased endogenous GPR120 expression in pig kidney cells (PK). Furthermore, when endogenous C/EBPß was knocked down, GPR120 mRNA and protein levels were decreased. The stimulatory effect of C/EBPß on GPR120 transcription and its ability to bind the transcription factor-binding site were confirmed by luciferase, ChIP, and EMSA. Moreover, the mRNA and protein expression levels of C/EBPß were induced by high fat diet feeding. Taken together, it can be concluded that C/EBPß plays a vital role in regulating GPR120 transcription and suggests HFD-feeding induces GPR120 transcription by influencing C/EBPß expression.

[Polymorphism in exon 2 of pig FIT1 gene and its association with fat-deposition-related traits].

In pig industry, fat deposition related traits such as back fat thickness and fat rate are of great economic importance. Thus, research on genes related with fat deposition can offer many useful values theoretically and practically. Gene FIT1 (Fat-inducing transcript 1) plays an important role in packaging lipid droplets. Here, we used FIT1 gene as the candidate gene for fat deposition. Sequence comparison revealed that an insertion/deletion mutation occurred at 590~595 bp of the second exon. We then carried out PCR-SSCP analysis followed by association analysis in F2 "Large white xMeishan" resource family. In all the individuals tested, all Meishan pigs possessed the insertion, which was designated allele A, while most Large white pigs possessed the deletion and was named as allele B. Association analysis in F2 resource family showed that this site was highly associated with fat percentage (FP), 6-7 rib fat thickness (RFT), buttock fat thickness (BFT), leaf fat weigh (LFW), total internal fat weigh (TFW), and internal fat rate (IFR) (Plt;0.01). These results indicated that FIT1 gene may have some important values for application. Further and deep research is necessary for revealing more information on this gene in order to provide a new marker for molecular marker-assisted selection breeding.

Characterization of the porcine differentially expressed PDK4 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.

Identification of a differentially expressed gene, ACL, between Meishan x Large White and Large White x Meishan F1 hybrids and their parents.

ATP-citrate lyase (ACL), one of the lipogenic enzymes, catalyses the formation of acetyl-coenzyme A (CoA) involved in the synthesis of fatty acid and cholesterol. In pig, very little is known about the ACL gene. In this work, the mRNA differential display technique was used to analyse the differences in gene expression between Meishan and Large White pigs and the F1 hybrids of both direct and reciprocal crosses. Our results show that among the differentially expressed genes ACL is up-regulated in the backfat of the F1 hybrids. After cloning and analysing the full-length cDNA and the 870 bp 50-flanking sequence of the porcine ACL gene, a C/T mutation at position -97 bp upstream of the transcription site was detected. Luciferase activity detection showed that this mutation changed the transcriptional activity. In F1 hybrids, the heterozygous genotype CT was more frequent than the homozygous genotypes CC and TT. Real-time PCR analysis showed that in Meishan pigs, ACL mRNA expression was more abundant in individuals with genotype CT than in those with genotype CC or TT or in Large White pigs. These results indicate that the C/T mutation affects ACL mRNA expression, probably via the activator protein 2.

Association of MYF5 and MYOD1 gene polymorphisms and meat quality traits in Large White x Meishan F2 pig populations.

MYF5 and MYOD1 belong to the myogenic regulatory factor (MRF) gene family. They code for the basic helix-loop-helix transcription factors that play key regulatory roles in the initiation and development of skeletal muscle and the maintenance of its phenotype. In this work three single nucleotide polymorphisms (SNPs) in porcine MYF5 and one in porcine MYOD1 were detected in three pig breeds (Large White, Landrace, and Meishan) by means of a PCR-RFLP protocol. Analysis of the association of meat quality traits with the four polymorphisms in a series of three Large White x Meishan F2 populations, totaling 399 pigs, found: (1) MYF5 exon 1 Hsp92II polymorphism causing a Met --> Leu substitution was associated with intramuscular fat content (P = 0.04) and water moisture content (P = 0.0001) in the longissimus dorsi; (2) MYF5 exon 2 MspI polymorphism and an intron 1 HaeIII polymorphism, which were completely linked, were significantly associated with longissimus dorsi pH (P < 0.05); (3) MYOD1 intron 1 DdeI polymorphism was not significantly associated with any meat quality traits tested. Among these genetic variants (a novel SNP and three identified SNPs), our data suggested that the novel SNP of the MYF5 gene within exon 1 is valuable for pig breeding.

Quantitative trait loci for carcass traits on pig chromosomes 4, 6, 7, 8 and 13.

For 22 carcass traits, we identified 16 QTLs (based on data for pig resource population no. 214, including 180 F2 hybrids of 3 Yorkshire boars and 8 Meishan sows) and mapped them with the use of 39 microsatellite marker loci on chromosomes 4, 6, 7, 8 and 13. Five QTLs were highly significant (P < or = 0.01 at chromosome level): for skin weight (on chromosome 7 at SW1856 and on chromosome 13 at SW1495), skin percentage (on chromosome 7 between SW2155 and SW1856 and on chromosome 13 between SW1495 and SW520), and ratio of leg and butt to carcass (on chromosome 4 at SW1996). The remaining 11 QTLs were significant (P < or = 0.05 at chromosome level): for backfat thickness at shoulder, loin eye width, loin eye height, fat meat weight, lean meat weight, skin weight, bone weight, skin percentage, fat meat percentage, and ratio of lean meat to fat meat. The proportion of phenotypic variance explained by these QTLs ranged from 0.06% (QTL for loin eye width on chromosome 8 between SW1037 and SW1953) to 18.04% (QTL for ratio of lean meat to fat meat on chromosome 7 between SW252 and SW581). Seven of the QTLs reported here are novel.

NNAT and DIRAS3 genes are paternally expressed in pigs.

Although expression and epigenetic differences of imprinted genes have been extensively characterised in man and the mouse, little is known on livestock species. In this study, the polymorphism-based approach was used to detect the imprinting status of NNAT and DIRAS3 genes in five heterozygous pigs (based on SNP) of Large White and Meishan F(1) hybrids. The results show that both genes were paternally expressed in all the tested tissues (heart, liver, spleen, lung, kidney, stomach, small intestine, skeletal muscle, fat, uterus, ovary and pituitary). In addition, the NNAT gene had two transcripts in all tested tissues, which is consistent with its counterpart in man and cattle.

Cloning and identification of porcine SMPX differentially expressed in F1 crossbreds and their parents.

In order to investigate porcine heterosis on the molecular basis, Large White (L), a European purebred, and Meishan (M), a Chinese indigenous purebred, were hybridized directly and reciprocally to produce F1 hybrids, Large WhitexMeishan (LM) and MeishanxLarge White (ML) pigs. Using mRNA differential display, we found an expression sequence tag (EST) differentially expressed in F1 hybrids and their parents, designated as EST55, which was homologous to human and murine skeletal muscle protein (SMPX), and the full-length cDNA of porcine SMPX was cloned by the rapid amplification of cDNA end (RACE) method. Translation of the mRNA transcript revealed an open reading frame (ORF) of 86 amino acid residues encoding a nuclear location signal peptide, two overlapping casein kinase II phosphorylation sites and one N-glycosylation site with theoretical molecular weight of 9.3 kDa. Alignment analysis revealed that the deduced protein sequence shared 94%, 83% and 78% homology with that of its human, mouse and rat counterparts, respectively. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that it was expressed predominantly in skeletal and heart muscles, whereas at a moderate level in backfat, spleen, stomach and uterus tissues. Two single nucleotide polymorphism (SNPs), located in 5'- and 3'-untranslated region (UTR), respectively,were identified by PCR and sequencing. Phylogenetic tree and the secondary structure prediction were also performed. The possible relationship between porcine SMPX and heterosis was discussed.

Molecular cloning and functional analysis of MRLC2 differential expressed in MeishanxYorkshire F1 crossbreeds and their parents, Meishan pigs.

In order to detect the molecular basis of heterosis in pigs, suppression subtractive hybridization was carried out to investigate the difference in gene expression in the Longissimus dorsi muscle tissues between MeishanxYorkshire F1 crossbreeds and their parents, Meishan pigs. The swine myosin regulatory light chain 2 (MRLC2) gene differentially expressed between the crossbreeds and the purebreds was isolated and identified using semi-quantitative reverse transcriptase polymerase chain reaction and its complete cDNA sequence was obtained using the rapid amplification of cDNA ends method. The nucleotide sequence of the gene is not homologous to any of the known porcine genes. The sequence prediction analysis reveals that the open reading frame of this gene encodes a protein of 172 amino acids containing the putative conserved domain of the EF-hand superfamily. This predicted amino acid sequence of porcine MRLC2 protein exhibits 99%, 98%, 98%, 98% and 97% identity with that of cattle, human, dog, rat and mouse, respectively. The homology analysis revealed that the MRLC2 protein was very much conserved in evolution. The tissue expression analysis indicated that the swine MRLC2 gene is highly expressed in muscle, fat, heart, liver, spleen, lung, kidney, stomach, small intestine, ovary and testis, but not expressed in pancreas.

Identification of a differentially expressed gene PPP1CB between porcine Longissimus dorsi of Meishan and Large WhitexMeishan hybrids.

To study the molecular basis of heterosis, suppression subtractive hybridization was used to investigate the differences in gene expression between porcine Longissimus dorsi of F1 hybrids Large WhitexMeishan and their female parents Meishan. From two specific subtractive cDNA libraries, the clones selected by reverse Northern high-density blot screening were chosen to clone full-length cDNA by rapid amplification of cDNA ends. An expression-upregulated gene for Meishan skeletal muscle, designated protein phosphatase 1, catalytic subunit, beta isoform (PPP1CB), was identified. Porcine PPP1CB contains an open reading frame encoding 327 amino acid residues with 13 and 1763 nucleotides in the 5' and 3' untranslated regions, respectively. A DNA fragment of 721 nucleotides was amplified and a mutation that creates/disrupts a restriction site for endonuclease RsaI was found. The derived amino acid sequence of PPP1CB has high homology with the PPP1CB of three species, Mus musculus (99%), human (99%) and mouse (100%). The tissue expression analysis indicated that the swine PPP1CB gene is generally expressed in most tissues. The possible role of PPP1CB and its relation to porcine heterosis are discussed.

cDNA cloning, sequence analysis of the porcine LIM and cysteine-rich domain 1 gene.

LIM domain proteins are important regulators in cell growth, cell fate determination, cell differentiation and remodeling of the cell cytoskeleton by their interaction with various structural proteins, kinases and transcriptional regulators. Using molecular biology combined with in silico cloning, we have cloned the complete coding sequence of pig LIM and the cysteine-rich domain 1 gene (LMCD1) which encodes a 363 amino acid protein. The estimated molecular weight of the LMCD1 protein is 40,788 Da with a pI of 8.39. It was found to be highly expressed in both skeletal muscle and cardiac muscle. Alignment analysis revealed that the deduced protein sequence shares 86%, 91% and 93% homology with that of its human, mouse and rat counterparts, respectively. The LMCD1 protein was predicted by bioinformatics software to contain a novel cysteine-rich domain in the N-terminal region, two LIM domains in the C-terminal region, nine potential protein kinase C phosphorylation sites, seven casein kinase II phosphorylation sites, a tyrosine kinase phosphorylation site, seven N-glycosylation and N-myristoylation sites and a single potential N-glycosylation site, which is similar to the protein's human counterpart. Phylogenetic tree was constructed by aligning the amino acid sequences of the LIM domain from different species. In addition, four base mutations were detected by comparing the sequences of Large White pigs with those of Chinese Meishan pigs. The G294A mutation site was confirmed by polymerase chain reaction-single-strand conformation polymorphism analysis. Its allele frequencies were studied in five pig breeds.

[Sequence and polymorphism analysis of porcine hormone-sensitive lipase gene 5'-UTR and exon I].

Hormone-sensitive lipase (HSL) is the key enzyme responsible for the mobilization of free acids from adipose tissue, and it is also the most important enzyme that affect fat deposition. In this paper, the porcine hormone-sensitive lipase gene 5'-UTR and exon I were sequenced. The sequence number in GenBank are AY332499, AY332497, AY332504, AY332505. A GC-CG in the DNA sequence -13 - -12 bp of porcine HSL gene 5'-UTR was detected between Duroc, Meishan, Qingping pig, Largewhite and Landrace. A G-->A missense mutation was detected in HSL gene exon I of different pig breeds. The characterization of the BsaH I PCR-RFLP polymorphism in exon I of the porcine HSL gene of different breeds and "Largewhite x Meishan" F2 group was analyzed. By association analysis between BsaH I PCR-RFLP polymorphism and GG, GG, AA genotypes of HSL gene exon I, a significant difference of pig eye area was found between AG and GG genotypes (P<0.05) in F2 group.

[Mapping quantitative trait loci for meat quality trait in a Large White x Meishan cross].

To search for the chromosome regions for quantitative trait loci (QTL) affecting meat quality in pigs, a three-generation resource family was developed in China using three Large White grand sires and seven Meishan grand dams. A total of 147 F2 progenies derived from two populations in 1998 (n = 81) and 2000 (n = 66) were phenotyped for meat quality. All animals were typed for 48 microsatellite markers covering six chromosomes: 1, 2, 3, 4, 6 and 7. Linear model and least square analyses were used for interval mapping meat quality in jointed population and single population, permutation for empirical threshold. The strongest linkage at chromosome-wide level (P < 0.01) and genome-wide level (P < 0.05) on chromosome 4 affecting intramuscular fat (IMF) QTL from the population in 2000 was detected with explained phenotypic variance of 5.24%, and Meishan's QTL increased the intramuscular fat content. There was a suggestive QTL for IMF near the threshold of chromosome-wide in the same region in jointly population. The QTL for pH value in m. Semispinalis Capitis and m. Biceps Femoris were located on chromosomes 1 and 3, respectively. One and three QTL affecting WHC reached the thresholds of chromosome-wide level in the populations of 1998 and 2000, respectively. In the population of 1998, QTL for moisture content at chromosome-wide level was on SSC6 and in jointed populations were on SSC2, 6 and 7. There were imprinting effects in moisture content QTL, and Meishan and Large White pigs all had favorable effects influencing moisture on different chromosomes.

[Mapping quantitative trait loci for fat deposition in carcass in pigs].

One of the major determining factors in the price of market hogs today is backfat depth. Therefore, identification of regions of the genome affecting this trait is necessary. Gene-mapping technologies have provided scientists the necessary reagents to conduct genomewide searches for genes affecting any phenotype determined in part by the genetic makeup of the animal. Over the past few years, several experimental crosses have been used to detect quantitative triat loci (QTL) for fatness and meat quality traits in pigs. In an intercross between Large White and Meishan pigs, 81 F2 progenies in 1998 were slaughtered and phenotyped for fat deposite traits. F2 animals, their parents, and their grandparents were typed for molecular markers covering chromosomes (SSC) 1,2,3,4,6 and 7, since previous studies had revealed QTL affecting fatness traits on these chromosomes. Linear model and least square analyses were used for mapping these traits. Furthermore, a QTL accounting for imprinting effects was used. A total of 14 QTL at chromosome-wide level were detected on SSC1, 3-4, 6 and 7 for 12 fatness traits. There were evidences for imprinting effects on SSC7 for four traits at 60 cM and nearby. There were QTL for average backfat thickness (BFT) on SSC1, 4 and 7. Among them, BFT QTL on SSC1 and 7 may be the common locus for Sus scrofa. QTL for internal fat percentage was on SSC7 and QTL for average sidefat on SSC6.

[Detection of quantitative trait loci for growth in large white x Meishan intercross].

The development of molecular biology techniques and the application of these techniques to farm animals have progressed rapidly and have opened new vistas for investigators wishing to identify genes that control quantitative traits. Now that a comprehensive map has been developed for the porcine genome, genomic scans to detect quantitative trait loci (QTL) can begin. In order to locate the genetic regions in the swine genome that are responsible for economically important traits, a resource population was developed by intercross with three Large White and seven Meishan pigs. In subsequent generations, 66 F2 offsprings in 2000 were recorded for four growth traits including birth weight (BWT), body weight at 60 day (WT60), average daily gain from birth to 60 day (ADG1) and average daily gain from 60 day to the end of test (ADG2), and genotyped for 48 microsatellite markers spanning six chromosomes. Association analyses were performed using interval mapping by regression under an outbred line-cross model on chromosomes 1, 2, 3, 4, 6 and 7. The F threshold values were determined by permutation. A total of 12 QTL were detected at suggestive level for the four traits evaluated in this study. Of the 12 suggestive QTL, 3 and 1 QTL were significant at the chromosome-wise and genome-wise levels. There was a QTL for ADG2 at genome-wide level on chromosome 4 explained additive variance 2.19%. A chromosome-wide QTL affecting ADG1 and WT60 were detected on chromosomes 2 and 1 explained additive variance 0.01% and 26.01%, respectively.

[Polymorphism of porcine myostatin gene].

Myostatin(MSTN) gene is expressed specifically in developing and mature skeletal muscle, and the expression products of MSTN gene inhibits muscle growth and differentiation. The polymorphism of the porcine MSTN gene was researched by PCR-SSCP. The SSCP was found in the MSTN gene exon 2 and exon 3. It showed three genotypes (CC, CT, TT) in exon 2 and two genotypes (AG, GG) in exon 3 in Large White pigs. The relationship between polymorphism in exons 2 and 3 and productive performance was analysed. Variance analysis showed that there was no significant relationship between the polymorphism in exon 2 and productive performance. But t test showed that there was a significant relationship between the polymorphism in exon 3 and back fat thickness (P < 0.05), there was also a relationship between the polymorphism in exon 3 and lean meat percentage, but not significant (P > 0.05). The fragments with SSCP polymorphism in exon 2 and 3 were sequenced, the sequencing results showed that there were two single nucleotides mutation, i.e. A-->G at 1008 (exon 3) and G-->T at 480 (exon 2). Two mutations did not change the amino acid but brought an Apa I site in exon 3, and PCR-RFLP molecular marker technique was established with Apa I.

[Construction of linkage map of chromosomes 1 and 3 in large white x Meishan reference family].

A three-generation family of pigs has been constructed by using three Large White boars and seven sows of Meishan pigs as parents. In this family, five F1 males and twenty-three F1 females were intercrossed to generate 147 F2 offspring. According to the pig linkage map of USDA-MARC, eight and nine microsatellite markers selected on chromosomes 1 and 3 were chosen to span the entire chromosomes. The members of this family were genotyped. The characterization of these microsatellites was shown that they were polymorphic and could be used to construct linkage map and detect quantitative trait loci (QTLs). Linkage analyses were performed using the CRI-MAP software package. The lengths of the sex-averaged linkage map were 182.3 cM and 180.2 cM on chromosomes 1 and 3, respectively. There were some differences between the linkage maps in this study and of USDA-MARC. The linkage map of chromosome 1 in female was found to be shorter than in male, and the contrary was on chromosome 3.