解析猪miR-22前体序列上游G>A突变位点影响猪肉色变异的分子机理

肉色是影响猪肉品质的主要因素之一。现有研究已鉴定出众多猪肉色相关QTL区域和SNP标记，但已鉴别的真正功能性的因果突变位点较少。申请人前期研究发现，miR-22可影响成肌细胞增殖与分化且在猪不同肉色肌肉中差异表达，其前体序列上游存在NC_010454.4:g.47913559 G>A突变位点与肉色a值（红度值）极显著相关，该突变经预测可调控miR-22的表达，且miR-22在不同基因型个体肌肉中差异表达。本研究推测该突变位点通过影响miR-22表达参与调控猪肉色，并拟先利用CRISPR/Cas9技术对猪骨骼肌细胞中该位点进行定点编辑，探究其对miR-22表达的影响；再通过研究miR-22靶向猪肉色相关差异表达基因ELOVL6调控肌肉细胞脂肪酸组成、脂肪酸氧化、线粒体功能及肉色标志基因变化的特征，揭示该位点影响猪肉色的分子机制。研究成果可为我国地方品种猪肉色性状早期选育提供新的遗传标记。

Meat color is one of the main factors that determine meat quality. Current researches have successfully identified a series of genomic regions and SNP markers related to the pig meat color trait, whereas the work on identifying causative mutations of meat color trait yet to be fully developed. Our previous study found that miR-22 could influence myoblast proliferation and differentiation. And in different color types of muscle in pigs, the expression of miR-22 was different. A NC_010454.4: g.47913559 G>A mutation site was discovered in the upstream of miR-22 precursor sequence and was significantly associated with pig meat color a* value (redness value). miR-22 was differentially expressed in muscle from pigs of different genotypes. Therefore, we speculated that this site might regulate pig meat color by affecting the expression of miR-22. In this study, CRISPR/Cas9 technology will be first used to edit this mutation site in porcine skeletal muscle satellite cells to fully verify its effect on the expression of miR-22. Furthermore, the molecular mechanisms of miR-22, targeting the meat color related gene ELOVL6, in the regulation of muscle fatty acid composition, muscle fatty acid metabolism, mitochondrial function and meat color marker gene expression in porcine skeletal muscle satellite cells will be explored. These results will reveal the role of NC_010454.4: g.47913559 G>A in affecting meat color variation. The research results will provide a new genetic marker for the early meat color breeding in our native pig breeds.