固始鸡×安卡鸡资源群IFM含量表型变异相关基因及其转录后调控机制研究

Intramuscular fat（IMF）contents in chicken meat is regarded a important trait related to meat quality. To unearth the excellent trait in Gushi chicken, Gushi×Anak chicken resource population was constructed. Our previous research shown that the IMF content of breast muscle in the resource population was a high level of phenotypic variation. But its molecular regulation mechanism is not clear. It is known that miRNAs participate in the regulation of many biological processes related to fat deposition via targeting the adipogenesis related genes. Therefore, in this study, Gushi×Anak chicken resource population will be used as experimental material. For the breast muscle and liver samples of the individual with distinct IMF contents in pectoral muscle tissue，proteomic profile and miRNA expression profile will be constructed using iTRAQ(isobaric tags for relative and absolute quantitation) technology and Solexa sequencing technology to identity the genes and miRNAs involved in the phenotypic variation of IMF content, respectively. Then, the microRNA-target pairs relating the phenotypic variation of IMF content are identified through the integrative proteomic profile and miRNA expression profile data together with computational approaches. And then, the network diagram of miRNA-gene target interactions is constructed to screen the core microRNA-target pairs involved in the phenotypic variation of IMF content. Final, the role of the core microRNA-target pairs during adipogenesis in chicken is tested by experimental validation in cell and living level. In conclusion, a deeper understanding of post-transcriptional control mechanism relating chicken IMF deposition will be achieved through all above research. The results of this study will provide the rationale base for elucidating the molecular mechanism of fleshy trait development involved in IMF content in the domestic chicken (Gallus gallus). It should be beneficial to protect and exploitate the Gushi-chicken germplasm resource.

肌内脂肪（IMF）含量与鸡肉品质密切相关。为发掘固始鸡优异性状，构建了固始鸡×安卡鸡资源群，该资源群胸肌IMF含量高度变异，但其分子调控机制尚不清楚。已知miRNA通过靶作用于脂肪形成相关基因而参与脂肪沉积的许多生物学过程调控。为此，本研究以固始鸡×安卡鸡资源群为素材，采用iTRAQ质谱定量技术和Solexa测序技术，构建胸肌不同IMF含量个体肝脏和胸肌样品的蛋白质组谱和miRNA表达谱，鉴定IMF含量变异相关基因和miRNA；通过miRNA和蛋白质组谱整合分析，鉴定IMF含量变异相关miRNA-靶基因对；构建miRNA-靶基因互作网络，筛选IMF含量变异相关核心miRNA-靶基因对；细胞水平和在体水平验证核心miRNA-靶基因对对鸡脂肪形成的影响，最终揭示鸡IMF沉积的转录后调控机制，为阐释地方鸡IMF含量相关肉质性状形成的分子机制提供理论依据，从而有利于固始鸡种质资源的保护与利用。

本项目围绕固始鸡F2资源群肌内脂肪（IMF）含量表型变异的分子调控机制解析，开展了以下研究：①以胸肌组织样品和肌内脂肪细胞为试验素材，采用iTRAQ质谱定量技术和Solexa测序技术，鉴定了固始鸡IMF沉积相关基因和miRNA；②在细胞水平，研究了miR-15a、miR-223、miR-128-3p和miR-27b-3p在固始鸡IMF沉积中的作用与调控机制；③评价了鸡miR-21、miR-92、miR-124a-3p和miR-125b的组织表达谱和生物学功能。取得了以下重要结果：.（1）IMF沉积相关基因：一是基于蛋白组谱数据，分别从固始鸡F2资源群不同IMF含量个体胸肌和肝脏组织中鉴定到2,284和3,692种蛋白。二是基于6、14、22和30周龄固始鸡胸肌转录组谱和IMF相关性状表型数据，通过WGCNA分析，鉴定到6个IMF性状相关特异性转录模块和98个hub基因。三是通过Ribo-Zero RNA测序，由鸡肌内和腹部脂肪衍生前脂肪细胞中鉴定到2,039个差异表达基因。.（2）IMF沉积相关miRNA：一是基于6、14、22和30周龄固始胸肌组织的四个小RNA文库，共鉴定到388种已知miRNA和31种新miRNA。二是基于肌内前脂肪细胞和诱导后10天的分化脂肪细胞的小RNA文库，分别鉴定到300和359种已知miRNA。.（3）miR-15a促进鸡肌内前脂肪细胞分化机制。根据我们的试验结果，miR-15a可通过直接靶作用于ACAA1、ACOX1和SCP2并抑制它们的表达而降低PPAR信号通路中的脂肪酸氧化作用，从而增加了鸡肌内脂肪细胞中脂肪酸的含量。蓄积的脂肪酸，进一步促进了PPARs的转录激活，进而增加了下游脂肪生成基因的表达，最终导致鸡肌内前脂肪细胞的分化。.（4）miR-128-3p和miR-27b-3p在鸡肌内脂肪细胞分化过程的调控机制。基于我们的研究，在IMFNCR序列上存在一个miR-128-3p和miR-27b-3p的结合位点；PPARG是鸡miR-128-3p和miR-27b-3p的一个直接靶基因。IMFNCR可结合隔离miR-128-3p和miR-27-3p，提高PPARG表达，从而促进肌内脂肪细胞分化。.本项目所得研究结果，拓展了鸡IMF沉积相关基因和miRNA的数量，为阐释地方鸡IMF含量相关肉质性状形成分子机制提供了有价值的资源。