Identification of a potential functional single nucleotide polymorphism for fatness and growth traits in the 3'-untranslated region of the PCSK1 gene in chickens

Abstract

Prohormone convertase 1/3 is a serine endoprotease belonging to the subtilisin-like proprotein convertase family that is encoded by the () gene, and its major function is the processing and bioactivation of the proproteins of many kinds of neuroendocrine hormones, including insulin, cholecystokinin, and adrenocorticotropic hormone. The results of our previous genomewide association study indicated that the gene might be an important candidate gene for fatness traits in chickens. The objectives of this study were to investigate the tissue expression profiles of gene and to identify functional variants associated with fatness and growth traits in the chicken. The results indicated that mRNA was widely expressed in various tissues, especially neuroendocrine and intestinal tissues. Of these 2 tissue types, mRNA expression in lean males was significantly higher than in fat males. A SNP in the 3' untranslated region of (c.*900G > A) was identified. Association analysis in the Arbor Acres commercial broiler population and Northeast Agricultural University broiler lines divergently selected for abdominal fat content (NEAUHLF) population showed that the SNP c.*900G > A was associated with abdominal fat weight, abdominal fat percentage, BW, metatarsus length, and metatarsal circumference. In the 5th to 19th generation (G to G) of NEAUHLF, the allele frequency of c.*900G > A changed along with selection for abdominal fat content. At G, allele G of c.*900G > A was predominate in the lean line, whereas allele A was predominate in the fat line. Functional analysis demonstrated that allele A of c.*900G > A reduced mRNA stability and consequently downregulated gene expression. These results suggested that c.*900G > A was a functional SNP for fatness and growth traits in the chicken. The results of this study provide basic molecular information for the role of gene in avian growth and development, especially obesity.

Figure 1.

Characterization of the tissue expression pattern of the chicken proprotein convertase subtilisin/kexin type 1 (PCSK1) gene in transcriptional level in 7 wk of age lean and fat male broilers of the Northeast Agricultural University broiler lines divergently selected for abdominal fat content population. TATA box binding protein (TBP) was used as an internal reference gene.

Figure 2.

Difference of proprotein convertase subtilisin/kexin type 1 (PCSK1) mRNA expression levels between lean and fat male birds. TATA box binding protein (TBP) was used as an internal reference gene. The results were expressed as means ± SEM (n = 6 animals/group). *P < 0.05; **P < 0.01.

Figure 3.

The change of allele frequencies of SNP c.*900G > A along with the selection for abdominal fat content from the 5th to the 19th generation (G₅ to G₁₉) in the Northeast Agricultural University broiler lines divergently selected for abdominal fat content population. AFP = abdominal fat percentage.

Figure 4.

The effects of different alleles of c.*900G > A on luciferase activities and mRNA stability. (A) Relative luciferase activity was calculated as the ratio of synthetic Renilla luciferase (hRluc) activity vs. firefly luciferase (hFluc) activity. DF-1 cells were transfected with 1.0 µg of the psiCHECK-2 plasmids (PCSK1G/A-psiCHECK-2) in 12-well plates. Cells were harvested 32 h after transfection, and the activities of hFluc and hRluc were measured. The results were expressed as means ± SEM. **P < 0.01. (B) DF-1 cells were transfected with 1.0 µg of the psiCHECK-2 plasmids (PCSK1G/A-psiCHECK-2) in 12-well plates. Twenty-four hours after the transfection, the cells were treated with 5 g/mL actinomycin D (ActD). Ribonucleic acid was extracted at the indicated times. The amount of hRluc transcript remaining at each time point was measured by quantitative reverse-transcription PCR by the absolute quantitative method, and the percentage of remained hRluc transcript was calculated with the 0-h time point set to 100%. The linear regression equation was established according to RNA amounts at each time point, and the half-life was calculated according to the regression equation.