| In this study, we chose hypothalamus cDNA as template, the full-length cDNA sequence of porcine SIM1 gene was cloned. The 3720 bp porcine SIM1 cDNA sequence was obtained by RT-PCR and RACE techniques. Further analysis revealed an open Reading Frame (ORF) of 2301 bp which encoding a corresponding protein of 766 amino acid residues with a predicted molecular weight of 85.3 kDa and theoretical isolectric point of 7.29. The cDNA sequence contained a 5'untranslated region of 461 bp and a 3'untranslated region of 958 bp. The gene structure, protein structure and function of porcine SIM1 gene were analyzed and predicted using bio information software, the results showed that the cDNA sequence showed a high identity with cattle (92.8%), human (94.5%), chimpanzee (91.4%), rhesus monkey (94.4%). The corresponding protein contained the conserved functional domain of bHLH-PAS protein, and was evolutionarily conserved with that of mammals. The ortholog of porcine SIM1 is related to cattle counterpart.Real-time PCR technique was used to detect expression profile and developmental expression pattern in subcutaneous fat of porcine SIMl mRNA. The results showed that SIM1 gene showed ubiquitous distribution in all examined tissues including stomach, spleen, pancreas, heart, lung, kidney, subcutaneous fat, muscle, liver and hypothalamus, while particularly higher in kidney and muscle, the lowest level was detected in subcutaneous fat. The developmental expression pattern study was carried out in subcutaneous fat of Jinhua pig and Yorkshire, the results showed that SIM1 gene expression continuously increased during 20d-120d, then decreased at 150d. Expression levels were higher in Yorkshire at six stages. No significant differences were found within different stage in any of the two breeds.Western blot technique was used to detect expression profile of porcine SIM1 protein and developmental expression pattern in muscle. The results showed that SIM1 protein were detected in all seven examined tissues, while particularly higher in brain and hypothalamus, lowest level was detected in subcutaneous fat, protein expression profile was similar to that of mRNA level. Developmental expressionpattern study in Jinhua pig only found a trace amount of expression at 20d and 45d, and no expression was found in following stages. The same study in Yorkshire revealed a persistent increasing expression during 25d-120d. reached the peak at 120d, then dropped at 150d. The backfat thickness and intramuscular fat content of Jinhua pig was significantly higher than that of Yorkshire, we speculated that SIM1 gene expression was inversely proporational to fat-related traits. SIM1 might involve in Leptin-Melanocortin-oxytocin pathway, regulating fat metabolism by adjusting dietary intake and energy metabolism.Two novel SNPs C534T and T1677C were found in porcine SIM1 gene. Distribution pattern in pure breeds were detected using PCR-SSCP. The result showed that frequency of allele C was higher in foreign pigs than domestic variates in C534T locus, only TT genotype was found in foreign breed at T1677C locus. Genetic effect were calculated in PJF2 resoure population, the substitution of T534C showed an extremely significant effect on average backfat thickness, intramuscular fat content, intramuscular water content and ham lean weight (p< 0.01), furthermore, it was significantly associated with ham fat weight (p< 0.05). Allele T in this site was associated with higher fat proportion and lower ham lean production. The substitution of C1677T had extremely significant effect on average backfat thickness, ham temperature and ham fat weight (p< 0.01), significant effect was observed on loin eye area, ham pH and ham lean weight (p< 0.05). Allele T in this site significantly decreased fat content but increased ham lean weight and loin eye area.PCR-SSCP and sequencing techniques were used to analyze amplifications of PPARy2 exons, and two substitutions were found, including A175G in exon 1 and G876A in exon 5. Allele frequency and genotype frequency were detected within 8 pig breeds. At A175G, allele A had a high frequency (> 75%) in European pig breeds, especially fixed in Pietrain swine, and a relatively lower frequency in Chinese native breeds (< 67%). Interestingly, at G876A, allele A was found only in Jinhua pigs, and allele G was fixed in all breeds except for this one. Therefore, allele A appeared to be Jinhua-specific. Linkage disequilibria between the two SNPs A175G and G876A, we chose haplotype analysis to estimate genetic effect between PPARy2 and fat-related traits. The association analysis were done within the PJF2 population. PPARy2 gene haplotypes were found to be significantly associated with BFT at the shoulder and gluteus medius (p< 0.05); with significant effects observed on ham weight and BFT at the 6-7th ribs and last rib (p< 0.01). Pigs with H1H3 genotype had lower ham weight than those with other genotypes (p< 0.01). H2H3 genotype pigs had higher BFT at the 6-7th ribs, last rib and gluteus medius (p< 0.01), while for BFT at the shoulder, the H2H3 genotype had relatively higher thickness just slightly lower than that of the H1H3 genotype. We concluded that PPARy2 is candidate gene for porcine fat-related traits.Two SNPs, T211C and A448G were found by alignment of UCP3 gene cDNA sequence of Jinhua pig and Pietrain. Allele and genotype frequency were detected within eight pig breeds using PCR-RFLP, the results showed that T221C polymorphism was monomorphic in all Chinese native breeds, in which only CC genotype was found. The allele T exists in European breeds. At the A448G locus, allele G has higher frequency than allele A in all pure breeds, except for Pietrain. Shengxian Hua pig has fixed genotypes in both polymorphism loci. Genetic effects were calculated in PJF2 resoure population, T221C substitution has no significant effect on fat-related traits. The substitution of A448G showed an extremely significant effect on intramuscular water content, intramuscular fat content and loin eye area (p< 0.01), nearly significant effect was also found between A448G and ham pH (p= 0.072). Pigs with GG genotype had higher backfat thickness at shoulder, backfat thickness at 6-7th ribs and intramuscular fat content. Allele A in A448G site significantly improvesd fat-related traits. |
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