猪肌内脂肪沉积的lncRNA鉴定及其调控机制研究

Intramuscular fat (IMF) plays an important role in pork juiceness, tenderness and flavor. IMF content depends on the adipogenic differentiation of intramuscular adipocytes. Our previous studies showed that IMF deposition ability of Chinese native fat-type Bamei pig breed is significantly higher than abroad introduced lean-type Large-white pig breed. By the long non-coding RNA (lncRNA) sequencing of porcine subcutaneous fat (SCF) of above two types, we identified a large plenty of differentially expressed lncRNAs and found that lncRNA regulated the adipogenic differentiation of subcutaneous adipocytes. Although we first confirmed that lncRNA was implicated in porcine SCF deposition, its effect and regulatory mechanism on adipogenic differentiation of intramuscular adipocytes are still unknown. Based on above analysis, we hypothesize that lncRNA is involved in regulation of adipogenic differentiation of intramuscular adipocytes, and plays a key role in IMF deposition difference between fat-type and lean-type pigs. Therefore, in order to solve the scientific problem on lncRNA regulation of IMF deposition, we will select Large-white pig and Bamei pig as the experiment animals to explore lncRNA regulatory mechanism on adipogenic differentiation of intramuscular adipocytes, analyze the genetic correlation between lncRNA gene and IMF content, and illuminate mechanism on differentially adipogenic differentiation of two types of pig intramuscular adipocytes and differential IMF deposition. The results of this project will provide novel candidate genes on selection and breeding of porcine IMF trait.

肌内脂肪是影响猪肉多汁性、嫩度和风味的关键，肌内脂肪细胞分化聚脂的能力决定肌内脂肪含量。申请人前期研究发现我国优良地方品种脂肪型八眉猪肌内脂肪沉积能力明显高于国外商用瘦肉型大白猪；通过对两种类型猪皮下脂肪lncRNA测序，鉴定了大量差异表达的lncRNAs，并率先发现lncRNA调控皮下脂肪细胞分化聚脂，但lncRNA对肌内脂肪细胞分化聚脂的作用机制仍不清楚。基于此，申请人推测lncRNA涉及猪肌内脂肪细胞分化聚脂的调控，并可能对脂肪型和瘦肉型猪肌内脂肪沉积差异发挥重要作用。因此，本项目以肌内脂肪沉积和肉质性状差异显著的八眉猪和大白猪为素材，针对lncRNA调控猪肌内脂肪沉积的科学问题，在分子、细胞和个体水平，探索其调控肌内脂肪细胞分化聚脂的机制，解析lncRNA基因与肌内脂肪含量的遗传相关，阐明两种类型猪肌内脂肪细胞聚脂和肌内脂肪沉积差异的机制，为选育猪肌内脂肪性状提供新的候选基因。

我国生猪产业是畜牧业乃至农业的支柱产业。目前，我国猪肉产量和消费量均居世界第一。尽管猪肉产量已满足国内消费需求，但猪肉品质亟待改善。脂肪型八眉猪比瘦肉型大白猪的肌内脂肪含量更高，因而八眉猪肉质更加鲜嫩多汁。为探究猪肌内脂肪沉积的机制，本项目分离培养了八眉猪和大白猪肌内脂肪细胞，收集分化0，2，4和8天的细胞样，检测其lncRNA的表达差异。测序结果显示共有760个新鉴定且差异表达的lncRNA并发现lnc-000414抑制脂肪细胞增殖。根据差异表达程度及生物信息学分析，从中筛选出6个与肌内脂肪沉积相关的lncRNA，并对3个lncRNA进行与猪肌内脂肪含量遗传相关分析。后续对lncIMF1、lncIMF2、lncFAM、lncIMF4做深入研究。研究结果表明，表达丰度最高的lncIMF1可通过吸附miR-187抑制猪肌内脂肪细胞增殖，促进猪肌内脂肪细胞聚脂分化；品种间差异表达最显著的lncIMF2促进猪肌内脂肪细胞成脂分化；干扰差异表达的lncFAM可以显著下调猪肌内脂肪细胞分化及脂质分解基因的表达；干扰差异表达的lncIMF4可显著抑制猪肌内脂肪细胞增殖，同时还会抑制猪肌内脂肪细胞自噬，抑制脂解，进而促进肌内脂肪细胞脂质沉积。本项目研究成功构建了lncRNA调控猪肌内脂肪沉积的网络，并从肌内脂肪细胞的增殖，分化，自噬和凋亡等多个生物过程探究了lncRNA对猪肌内前体脂肪细胞脂质积累的作用与机制。本项目研究结果完善了非编码RNA对猪肌内脂肪沉积的调控，为探索我国地方猪品种肉质优良特性的非编码RNA调控提供了新的候选基因，也为优质瘦肉型猪品种的选育和健康优质猪肉生产提供科学依据。此外，本项目还从不同部位脂肪沉积，功能基因和非编码RNA多角度对猪地脂肪沉积和肌肉发育进行了深入研究，这些重要成果为以后从遗传角度运用生物技术手段改善猪肉品质提供了新的思路和理论依据。