鸡肉质风味性状差异表达基因、miRNA的筛选及鉴定

Abstract: The meat flavor traits is one of the most important economic traits of chicken,researching on chicken’s meat flavor traits is focused on one or more genes so far,thoroughly revealing the regulation of genes influencing meat flavor traits, provening genetic mechanism of quality chickens, has important significance to the development and utilization of Rose Cockscomb Chicken —this valuable genetic resources.In this study, through comparative analysis of the differences between Rose Cockscomb Chicken and Avian Broiler’s meat flavor traits, differentially expressed genes in muscle tissue and miRNA is detected by RNA - seq and small RNA sequencing technology, differentially expressed involving-genes and miRNA’s regulatory is verified in cellular and individual level, and meat flavor traits regulatory pathway is constructed through jointing analysis of miRNA-mRNA, on this basis, the involed new genetically CRISPR / Cas9 knockout vector is designed and the candidate gene’s function in meat flavor traits emerging is detected.The purpose of this study is to explore the regulating mechanism of related genes in the metabolic pathway,clear key genes and miRNA involved in it,which has important significance in promoting further understand of molecular mechanism in chickens’ meat flavor traits.

摘要：肉质风味性状是鸡重要的经济性状，目前对于鸡肉质风味性状的研究多集中在某个或某些基因的研究，深入揭示影响肉质风味性状基因的调控，探明优质鸡肉的遗传机理，对开发利用玫瑰冠鸡这一宝贵的遗传资源具有重要意义。本研究对玫瑰冠鸡和艾维茵鸡的肉质风味性状进行比较分析，拟利用RNA-seq和small RNA测序技术检测肌肉组织中差异表达基因和miRNA，对筛选到的差异基因及miRNA在细胞和个体水平验证其调控作用，通过miRNA-mRNA联合分析构建肉质风味性状调控通路，在此基础上对筛选的新基因设计CRISPR/Cas9敲除载体，体外检测候选基因在肉质风味形成中的功能。本研究旨在探讨肉质代谢通路中相关基因的调节机制，明确参与调控的重要基因和miRNA，对进一步了解鸡肉质风味性状的分子调控机制具有重要的促进意义。

玫瑰冠鸡是石河子大学动科院正在培育的地方品种，其桑葚状冠型巨大鲜艳，肉质细嫩，香气浓郁，极具食用和观赏价值。科宝鸡是快大型商品肉鸡，生长速度快、饲料转化率高等优点，但肉质风味较差。玫瑰冠鸡与科宝鸡是研究肉质性状差异的理想模型。肌内脂肪是影响肉品质性状的重要因素，其含量影响肉的嫩度、风味和多汁性等。项目通过对比分析两种鸡的肉品质性状，进而对胚胎期肌肉进行高通量测序，获得与IMF沉积相关的miRNA、lncRNA及其靶基因，研究其涉及的通路及功能，以期探明肌内脂肪对肌肉风味调控的分子机理。.（1）玫瑰冠鸡的总氨基酸、必需氨基酸总量、鲜味氨基酸总量均高于科宝鸡（P<0.05），玫瑰冠鸡的肌内脂肪、肌苷酸（IMP）和矫正肌苷酸（IMPc）含量均高于科宝鸡，玫瑰冠鸡的硫胺素、还原糖含量高于或者持平科宝鸡。.（2）构建玫瑰冠鸡、科宝鸡胚胎期肌肉miRNA及转录组文库，120μmol/L油酸可诱导鸡前脂肪细胞（ICP2）分化，APOA1是miR-1677-5p的靶基因，过表达miR-1677-5p显著下调APOA1的表达量。miR-1677-5p可抑制其靶基因APOA1的表达，参与鸡前脂肪细胞分化，抑制鸡前脂肪细胞增殖；BMP2是miR-1306-5p的靶基因，过表达miR-1306-5p显著下调BMP2表达量。miR-1306-5p通过靶向调控BMP2基因的表达抑制鸡前脂肪细胞的分化及增殖。.（3）鉴定了1100个已知lncRNA、13180个未知lncRNA和30252个mRNA。差异性分析获得132个差异表达lncRNA和2693个差异表达mRNA。获得lncRNA-ENSGALT00000046546全长序列，构建过表达载体和siRNA，转染ICP2细胞， lncRNA-ENSGALT00000046546正向调控AGPAT2基因的表达。过表达组ICP2细胞脂滴更密集，细胞增殖受到了抑制lncRNA-ENSGALT00000046546促进ICP2细胞甘油三酯的合成，且脂肪代谢相关基因PPARγ、C/EBPα、FAS、SREBP1和FABP4基因的表达量均有极显著增加（P＜0.01），干扰lncRNA-ENSGALT00000046546的表达时，ICP2细胞增殖受到的影响较小。研究结果为鸡肌内脂肪形成差异的细胞分子遗传机制奠定基础，对今后优质肉质性状的选育提供理论依据。