猪肌内脂肪沉积过程中miRNA调控机理研究

Fat deposition affects meat quality. In particular, the suitable amount of intramuscular fat can significantly improve the taste of meat, juicy, tenderness and taste, thereby enhancing the quality of meat. By elucidating the molecular regulation mechanisms of intramuslular fat deposition process of pig, it would provide the theoretical basis for the realization of artificial regulation on intramuscular fat deposition, improving meat quality, accelerating the progress of breed improvement. In the proposed project, Jinhua pigs and Landrace, with extreme difference of intramuscular fat formation, will be used as animal model. Next-generation sequencing technologies will be used to screen all differentially expressed miRNA and expressed genes involved in intramuscular fat deposition early process of the two breeds (at postnatal day 7), and explore related miRNAs and their possible target genes early stage of intramuscular fat deposition. miRNAs responsible for intramuscular fat deposition were confirmed by real time PCR method. The candidate target genes of miRNAs will be verified with the luciferase reporter assay. MiRNAs over-expressing and interference in porcine intramuscular adipocyte will be constructed to understand their effect on the target genes and fat metabolism during the formation of intramuscular fat deposition and the network between miRNAs and genes. This project will provide useful evidence to the regulation of pork quality.

肌内脂肪与肉的嫩度、多汁性和风味呈正相关，是影响感官肉品质的一个重要的决定因素。阐明猪肌内脂肪沉积过程中的分子机理，能够为实现人工调控肌内脂肪沉积、改善肉品质、加快品种改良进程奠定基础。本项目以肌内脂肪存在显著差异的金华猪与长白猪为素材，拟运用第二代高通量测序技术，筛选出猪肌内脂肪沉积早期（出生后7天）差异表达miRNA和差异表达基因，寻找肌内脂肪沉积早期相关的重要miRNA及其作用靶基因；采用实时定量PCR检测技术，验证肌内脂肪沉积早期相关的miRNA；构建双荧光素酶报告基因系统，验证肌内脂肪沉积早期相关miRNA的候选靶基因；在细胞水平上，通过构建1-2关键miRNA过表达或抑制表达载体，探索miRNA与其靶基因以及猪肌内脂肪沉积发育相关信号通路基因表达和脂类代谢间的作用关系。研究猪肌内脂肪沉积早期关键miRNA的作用，并构建其与基因间的调控网络，阐释其作用机制，为猪肉品质调控提供重要依据。

肌内脂肪与肉的嫩度、多汁性和风味呈正相关，是影响感官肉品质的一个重要的决定因素，为了揭示miRNA调控猪肌内脂肪沉积的分子机理，本项目以肌内脂肪存在显著差异的金华猪与长白猪为素材，运用第二代高通量测序与生物信息学技术，构建了金华猪与长白猪肌内脂肪组织的小RNA文库，筛选出了220个差异miRNA (21个上调，199个下调)、1632个差异表达基因(837个上调，795个下调)、同时还发现了29个差异表达circRNA (19个上调，10个下调)。本项目采用qPCR技术对其中的24个猪肌内脂肪沉积相关的差异表达miRNA与对应的靶基因进行了功能验证。此外，通过构建双荧光素酶报告基因系统进一步验证了猪肌内脂肪沉积相关的关键miRNA (ssc-miR-370)的候选靶基因FASN，从而初步阐明了猪肌内脂肪沉积miRNA的作用，构建其与基因间的调控网络。