CIDEA基因上游调控区变异对猪肌内脂肪性状的影响

Effects of variation in the upstream regulatory region of CIDEA gene on intramuscular fat trait in pigs

[目的]本文旨在鉴定诱导细胞死亡DNA片段化因子α样效应因子A(CIDEA)基因上游调控区内与猪肌内脂肪沉积相关的单核苷酸多态性(SNP)位点,并分析关联位点对基因表达的影响,为解析CIDEA基因对猪肌内脂肪性状的影响机制提供参考。[方法]通过对猪CIDEA上游调控区2 kb片段测序,挖掘出肌内脂肪含量相关的候选SNP位点,在杜洛克×长白猪×大白猪群体中对候选位点进行关联分析;通过荧光定量PCR检测关联位点不同基因型个体的CIDEA基因表达量和肌内脂肪标记基因脂肪酸结合蛋白4基因(FABP4)、脂肪酸结合蛋白3基因(FABP3)的表达量;双荧光素酶报告基因系统分析突变位点对CIDEA基因转录活性的影响。[结果]猪CIDEA基因上游调控区包含6个SNP位点,连锁分析后选择g.97268816 T>A、g.97269217 G>A和g.97269353 C>T 3个标签SNP位点进行关联分析,发现g.97268816 T>A位点中AA基因型个体的肌内脂肪含量显著高于TT基因型个体(P<0.05),而g.97269217 G>A和g.97269353 C>T位点中3种基因型个体之间的肌内脂肪含量差异不显著(P>0.05)。g.97268816 T>A位点中AA基因型个体的CIDEA基因表达水平显著高于TT基因型个体(P<0.05),而FABP4基因表达水平则显著高于TT基因型个体(P<0.01)和TA基因型个体(P<0.05)。双荧光素酶报告基因活性的结果显示AA型个体的荧光素酶活性极显著高于TT型个体(P<0.01)。[结论]CIDEA基因上游调控区g.97268816 T>A位点与猪的肌内脂肪含量显著相关,突变可以导致CIDEA基因的表达水平升高,进而影响猪的肌内脂肪含量。

[Objectives]This study aimed to identify single nucleotide polymorphism(SNP)locus of the upstream regulatory region of cell death-inducing DNA fragmentation factorα-like effector A(CIDEA)gene related to intramuscular fat(IMF)deposition,and analyze the effect of these loci on gene expression,thus providing the reference information for resolving the influence mechanism of CIDEA gene on intramuscular fat trait of pigs.[Methods]The candidate SNP loci related to IMF content were discovered by sequencing the 2 kb fragment of the upstream regulatory region of porcine CIDEA gene.Then,association analysis was performed between these SNP loci and IMF contents in a Duroc×Landrace×Large White pig population.Real-time quantitative PCR was used to detect the expression levels of CIDEA,fatty acid binding protein 3 gene(FABP3)and fatty acid binding protein 4 gene(FABP4)on different genotype individuals.Dual-luciferase reporter gene system was performed to analyze the effect of SNP loci on transcriptional activity of CIDEA gene.[Results]A total of 6 SNP loci in the upstream regulation region of porcine CIDEA were discovered.After linkage analysis,g.97268816 T>A,g.97269217 G>A,and g.97269353 C>T loci were selected for the further analysis.The association analysis showed that the IMF content of AA genotype individuals in g.97268816 T>A locus was significantly higher than that of TT genotype individuals(P<0.05).However,there was no significant difference on IMF contents among the different genotype individuals in g.97269217 G>A and g.97269353 C>T loci.By analyzing the mRNA levels of the individuals with different genotypes of g.97268816 T>A locus,we found that the expression levels of CIDEA in AA genotype individuals were significant higher than those of TT genotype individuals(P<0.05),whereas the expression levels of FABP4 in AA genotype individuals were significant higher than those of TT genotype individuals(P<0.01)and TA genotype individuals(P<0.05),respectively.Moreover,the data from dual-luciferase reporter system indicated that the luciferase activity of AA genotype individual was highly significant higher than those of TT genotype individual(P<0.01).[Conclusions]The g.97268816 T>A mutation in the upstream regulatory region of CIDEA gene was significantly associated with the IMF contents of pigs.This mutation could increase the expression level of CIDEA gene,and then affect the IMF content of pigs.