Ntn1基因及其内部拷贝数变异调控猪肌内脂肪含量的分子机制研究

Intramuscular fat (IMF) content is an important economic traits for pig. Till now, little research was focus on the effects of copy number variations (CNVs) on IMF. Our previous results of genome-wide association between IMF content and CNVs indicated that the CNV within Netrin-1(Ntn1) was significant associated with IMF. In validation research, the CNV was confirmed, and the DNA dosage of this CNV (Ntn1-CNV) was proved consistent with Ntn1 RNA expression and IMF content. In the research of human and mouse, adenosine a2b receptor (a2bR), which is one of the receptors of Ntn1 could inhibit adipogenesis. We inferred that increased Ntn1 protein may bind with a2bR, and the decreasing of a2bR may lead to less inhibition effects on adipogenesis. Thus, this progress finally influence IMF content. In order to validate the effect of Ntn1-CNV and explore the molecular mechanism of Ntn1-CNV on IMF formation. Following research will be conducted: (1) study of the breed specificity of Ntn1-CNV, and association study of Ntn1-CNV and IMF content in different population; (2) study of the effect of Ntn1-CNV on Ntn1 protein expression content, and the space-time distribution pattern of Ntn1-CNV; (3) study of the relationship between Ntn1 and a2bR and other up-and down- regulated genes. The implementation of this project will help us to explain the molecular mechanism of IMF formation and provide us some DNA markers and theoretic base to pig meat quality traits molecular breeding, and will give researchers some advice to their research on the structure variations which could affect economic traits.

猪肌内脂肪（IMF）含量具有重要经济价值，其相关拷贝数变异（CNV）研究却十分匮乏。申请人前期进行了猪IMF含量全基因组CNVs关联分析，发现神经生长因子1（Ntn1）基因内存在与IMF含量显著相关的CNV，且此CNV拷贝数与Ntn1表达量及IMF含量趋势一致。人与鼠中的研究表明，Ntn1的受体腺苷a2b受体（a2bR）可抑制脂肪生成。推测此CNV可通过影响Ntn1表达量来调控Ntn1与a2bR的结合，进而调节IMF生成。为确证此CNV对IMF的作用，解析其影响IMF的分子机制。申请人拟开展: （1）此CNV的品种特异性及群体关联研究；（2）CNV差异对Ntn1蛋白表达量的影响及时空分布规律研究；（3）CNV差异对以Ntn1为结点的IMF生成相关基因调控网络的影响研究。为有效开发分子标记，开展肉质性状分子育种提供理论基础，同时为从结构变异角度研究影响经济性状的遗传因素提供方法上的参考。

本研究进行了NTN1基因内拷贝数变异（CNV）在长白猪、东北民猪、藏猪、北京黑猪以及二花脸猪等群体中的分布及作用验证。分别利用QPCR及Western-blot方法验证了CNV 差异对NTN1 蛋白表达量影响及时空分布，确认了NTN1基因对IMF的作用。利用RNA-seq方法对不同IMF含量的个体肌肉基因表达量进行了分析，共找到21个差异表达基因，利用这些差异基因构建了以NTN1 基因为结点的IMF 生成相关基因调控网络。培养3T3及C2C12细胞，分别利用基因沉默，QPCR等方法对NTN1基因对上下游基因的调控作用进行了验证，并对NTN1基因对脂肪及肌肉细胞的增殖与分化的影响进行了研究，结果发现，NTN1可影响肌肉细胞的增殖和分化。