鸡IGFBP2基因3′UTR区1196C>A单核苷酸多态性的功能性鉴定及分析

Identification and Analysis of a Functional SNP 1196C>A in 3′UTR of Chicken IGFBP2 Gene

鸡IGFBP2基因3′非编码区(3′UTR)1196C>A单核苷酸多态性(SNP)与鸡的腹脂重和腹脂率显著相关.生物信息学分析显示,该SNP恰好位于gga-mi R-456-3p的一个潜在靶基因结合位点处,提示IGFBP2基因可能是gga-mi R-456-3p的一个靶基因,且该SNP位点可能影响gga-mi R-456-3p对IGFBP2基因表达的调控作用.为鉴定SNP 1196C>A是否为功能性SNP,本研究分别构建了包含该SNP位点C或A等位基因的双荧光素酶报告基因载体,比较分析这两个等位基因在鸡胚成纤维细胞系(DF1)和鸡前脂肪细胞中对报告基因活性和表达的影响;利用mi RNA mimics和inhibitor分析gga-mi R-456-3p对不同等位基因报告基因活性以及内源性IGFBP2表达的影响.结果发现,在DF1细胞和鸡前脂肪细胞中,A等位基因的报告基因活性和表达均显著高于C等位基因(P<0.05);gga-mi R-456-3p仅影响C等位基因的报告基因活性和表达,而对A等位基因的报告基因活性没有明显影响;gga-mi R-456-3p调控细胞内源性IGFBP2基因的m RNA和蛋白质表达.本研究证实IGFBP2基因是gga-mi R-456-3p的靶基因,其3′UTR区SNP 1196C>A是一个功能性SNP,它影响gga-mi R-456-3p对鸡IGFBP2基因的表达调控作用.本研究结果对于鸡的分子辅助选择育种及IGFBP2基因在脂肪沉积中调控机制的阐明具有重要意义.

We previously mapped a QTL significantly influencing the abdominal fat weight and the percentage of abdominal fat of chicken on chicken chromosome 7, and IGFBP2 is the only known gene located within this QTL region. We further found that the 1196C〉A, a single nucleotide polymorphism （SNP） in 3＇UTR of Chicken IGFBP2 gene, is significantly associated with chicken abdominal fat weight and percentage of abdominal fat. Bioinformatics analysis suggests that this SNP is located at a potential binding site of gga-miR-456-3p. We hypothesized that IGFBP2 may be a target gene of gga-miR-456-3p, and this SNP may affect the gga-miR-456-3p-mediated downregulation oflGFBP2. To test and decipher this hypothesis, in the present study we constructed the 3＇ UTR reporter vectors for the two individual alleles of the IGFBU2 SNP （1196C〉A）, and compared the reporter activity between the two individual alleles in both DF1 cells and chicken preadipocytes. Using the miRNA mimics and inhibitor of gga-miR-456-3p, we further assessed the effects of gga-miR-456-3p on the reporter activity of the two individual alleles of the IGFBP2 SNP and the endogenous IGFBP2 expression at mRNA and protein levels in DF1 cells. The reporter assay showed that in both DF1 cells and chicken preadipocytes, allele A had higher reporter activities at protein and mRNA levels than allele C, indicating that this SNP is a functional SNP. Further studies demonstrated that in DF1 cells, gga-miR-456-3p mimcs and inhibitor had effect on allele C reporter activity, but not on allele A reporter activity. Quantitative real-time RT-PCR and Western blot analyses showed that gga-miR-456-3p mimcs and inhibitor regulated the endogenous expression of chicken IGFBP2 gene at mRNA and protein levels. Taken together, our results demonstrated that IGFBP2 gene is a target gene of gga-miR-456-3p, and the SNP 1196C〉 A is a functional SNP. Our findings are of great significance to the marker-assisted selection for lean chicken and clarification of gene function and regulation of chicken IGFBP2 gene in chicken adipose development.