Molecular Characterization And Epigenetic Regulation Of Genes Involved In Fat Metabolism In Swine And Chicken

Fat metabolism is affected by many factors in livestock, including heredity, nutrition, endocrine secretion, environment and so on, which can inform complicated regulation network. Lipid is essential for the development of human and animals, and is also the very important composition in organism. However, too much fat deposition brings about many problems in swine and chicken. On account of this, the molecular characterization and epigenetic regulation of genes, which are involved in fat metabolism, were studied in swine and chicken here. And pig BMAL1 (pBMAL1) was analyzed with both molecular biology and bioinformatics methods in order to search major genes affecting fat metabolism, which can contribute to the molecular breeding of swine. In addition , the effects of betaine supplementation to diet on partial growth performance and carcass traits were analyzed in chicken, and thus to approach the effects of dietary betaine supplementation on expression and methylation of fat metabolism genes, which laid the foundation for further studying gene expression regulated by betaine and the molecular mechanism of methylation. It is of important significance for the breeding of high quality and high yield chicken.PartⅠ:In present study, we cloned and sequenced the cDNA and partial non-coding regions of pBMAL1 with degenerate primers designed according to the sequences of human and mouse BMAL1 gene, analyzed its expression in adipose tissues and other tissues and further mapped its position on porcine chromosome. The results indicated that pBMAL cDNA had 2073 bp, including 1878 bp open reading frame (ORF), which coded 626 amino acids including bHLH, PAS-A and PAS-B domains. They shared 94.36%, 89.85%, 89.79% identity in coding region nucleotide and 99.20%, 98.24%, 97.92% in amino acid with the sequences of human, mouse and rat BMAL1, respectively. Two splice-variants were found in Landrace pig and Laiwu Black pig backfat. And the expression of pBMAL1 was detected in five adipose tissues of Landrace pig,with the highest expression level in mesentery fat tissue and the lowest in caul fat tissue, but the expression levels were not significantly different among them (P>0.05). And also the expression of pBMAL1 was detected in 14 tissues of adult Laiwu pigs, with the highest in backfat and the lowest in eye muscle. Furthermore, the pBMAL1 gene was mapped SSC 2p11-q21 with a whole genome porcine radiation hybrid (RH) panel (IMpRH).PartⅡ:This experiment was conducted to determine the effect of dietary betaine supplementation on partial growth performance and carcass traits in broiler and laying hen; the expressions of LPL, PPARγ, A-FABP, FAS, Adiponectin, BMAL1 and CLOCK were analyzed by semi-quantitative RT-PCR method. And the methylation status of LPL and PPARγpromoters were examined by bisulfite sequencing.Our results showed that the 0.1% of betaine addition to the diet showed no significant effect on average daily gain, percentage of abdominal fat and the ratio of liver to body weight in broiler (P>0.05). However, the addition 0.08% betaine to the diet decreased the ratio of liver to body weight by 15.66% compared with the control diet (P<0.05) in 165 d laying hen. Similarly, the daily average laying rates were increased by 26.18% (P<0.05) in 180 d laying hen by 0.08% betaine supplementation. Moreover, there were no significant differences on other traits by the addition different dosage of betaine to the diet during two test stages (P>0.05).For broiler, the addition of 0.1% betaine to the diet decreased significantly A-FABP mRNA expression level in 56 d and LPL mRNA expression level in 66 d of subcutaneous adipose tissue (P<0.05), respectively. Also, the FAS mRNA expression level of 0.1% betaine treatment groups were extremely significantly decreased compared with that of control groups in 56 d and in 66 d (P<0.01), respectively. Nevertheless, no significant differences were found in LPL mRNA expression in 56 d and A-FABP mRNA expression in 66 d by the same betaine supplementation (P>0.05), and neither 56 d broiler nor 66 d broiler of PPARγ, Adiponectin, BMAL1 and CLOCK mRNA expression levels were significant differences by the same betaine supplementation (P>0.05).For laying hen, semi-quantitative RT-PCR results indicated that LPL mRNA expression level in 180 d for betaineⅡgroup (0.06% betaine addition) was extremely significantly lower than those of the control group and betaineⅢgroup (0.08% betaine addition) (P<0.01); and FAS mRNA expression level in 180 d for betaineⅢgroup was extremely significantly higher than those of the other groups (P<0.01); and also both Adiponectin mRNA in 165 d and BMAL1 mRNA in 180 d for betaineⅢgroups were significantly higher than those for controls (P<0.05). The expression levels of other genes did not reach significance among groups in different test stages (P>0.05).Bisulfite sequencing analysis results revealed that LPL promoter region was hypomethylated in subcutaneous adipose tissue of 66 d broiler; percentages of global DNA methylation in control and betaine groups were 4.55% and 2.77%, respectively. However, PPARγ5′upstream regulatory region was in hypermethylation status in subcutaneous adipose tissue of 56 d broiler. And the global DNA methylation levels of PPARγdisplayed 76.7% and 71.7% in control and betaine groups, respectively. The DNA methylation level of LPL and PPARγwere not affected by betaine supplement in broiler subcutaneous adipose tissue (P>0.05).Similar observations were made for 180 d laying hen, in which the LPL promoters was hypomethylated. The global DNA methylation levels were 3.20%, 4.31%, 1.83% and 3.65% among control and betaineⅠ(0.04% betaine addition),ⅡandⅢgroups, respectively, and no significant differences were found among groups (P>0.05). And, PPARγ5′upstream regulatory region was also hypermethylated in subcutaneous adipose tissue of 180 d laying hen. The global DNA methylation status indicated 89.6%, 85.7%, 85.7% and 71.2%, respectively. And the difference of methylation levels was significant between control and betaineⅢgroup (P<0.05).