Abstract：The apolipoprotein family plays an important role in the transport and metabolism of lipids and cholesterol in the body. The apolipoprotein B (ApoB) plays an important role in lipid metabolism. Previously it was found that the genotypes of chicken (Gallus gallus) ApoB g.-112A>G (single nucleotide polymorphism, SNP) were significantly associated with abdominal fat weight and abdominal fat percentage in chickens. To determine whether the ApoB gene g.-112A>G is a functional SNP, we conducted the following research work: Association analysis between different genotypes and plasma biochemical markers, body weight and abdominal fat traits; The mRNA expression levels of the 3 ApoB genotypes in the liver and small intestine of chickens were detected and compared; The luciferase reporter gene vector containing different alleles in the promoter region of chicken ApoB gene was constructed and transfected into chicken fibroblast cell (DF1) and human hepatoma (HepG2) cells respectively; The possible mechanism of action of the ApoB gene g.-112A>G was predicted and analyzed by on-line bioinformatics software. It was found that the A allele could change the TBP and C/EBPα transcription factor binding sites in the chicken ApoB gene promoter region. It could be assumed that the g.-112A>G could influence the promoter activity of ApoB gene, which in turn affected gene expression. To confirm this hypothesis, the effect of pCMV-TBP and pCMV-C/EBPα on the activity of the dual-luciferase reporter gene of different alleles was analyzed in DF1 and HepG2 cells. The major results as follows: (1) Association analysis showed that genotypes of g.-112A>G in ApoB gene were significantly associated with high-density lipoprotein (HDL) (P＜0.01), and significantly associated with HDL/low-density lipoprotein (LDL) and Body Weight (BW) (P＜0.05), and closely associated with Triglyceride (TG)(P=0.0549), Total Cholesterol (TC)(P=0.0926) and Abdominal Fat Weight (AFW) (P=0.0805). Multiple comparisons showed that HDL, HDL/LDL, and BW were significantly higher in individuals with GG genotype than those with AG and AA genotypes (P＜0.05). (2) qRT-PCR results showed that the ApoB gene expression level of AG genotype was significantly lower than that of AA genotype and GG genotype in both chicken liver and small intestine (P＜0.05). (3) Bioinformatics predictions revealed that the A allele could change the TATA-box binding protein (TBP) and CCAAT/enhancer binding protein alpha (C/EBPα) transcription factor binding sites in chicken ApoB gene promoter region. (4) The study successfully constructed reporter gene vectors containing different alleles of the chicken ApoB gene g.-112A>G, and reporter gene activity analysis indicated that there were significant differences in the reporter gene activity of the A and G alleles (P＜0.05), and the reporter gene activity of the A allele was significantly higher than the G allele. (5) TBP eukaryotic expression vector pCMV-TBP was successfully constructed. pCMV-TBP and pCMV-C/EBPα (kept in the lab) were co-transfected with reporter vectors of different alleles. The results showed that transcription factors TBP and C/EBPα promoted luciferase activity of the A allele more strongly than that of the G allele (P＜0.05). The above results suggested that ApoB gene g.-112A>G might be a functional SNP locus, and it could be preliminarily identified as a functional molecular marker for quality chicken breeding. The present study provides experimental data for further exploration of the function of ApoB gene in fat development.
