【作者】 赵春平；

【导师】 昝林森；

【作者基本信息】 西北农林科技大学 ， 动物遗传育种和繁殖， 2012， 博士

【摘要】 目前，对于遗传因素影响牛肉品质的研究报道较多，但是关于环境因素和外界刺激影响牛肉品质的研究报道相对较少，特别是关于牛机体在遭受强烈刺激或者应激状态下肉质特性发生变化的研究鲜有报道。本试验以安格斯牛为材料，探索因外科手术引起的创伤性强烈应激对牛肉肉质性状的影响，期望阐明应激状态下牛肉食用品质的变化规律，为强烈刺激引起牛肉品质变化提供一些证据支持，并进一步探索内在的分子生物学机制。具体结果如下：（1）强烈应激引起牛肉品质显著变化。牛背最长肌的剪切力由对照组的5.040kg升高至试验组的13.366kg，即牛肉嫩度极显著降低（P<0.01）。同时肌肉中脂肪酸含量发生了显著变化。其中肉豆蔻酸、棕榈酸、棕榈油酸、硬脂酸和十八烯酸的含量在试验组中极显著升高（P<0.01）。应激组内不同个体应激反应的程度不同，因而肉质性状变异较大，应激组内剪切力分布为5.805～20.70kg。（2）利用Agilent基因芯片比较应激组表型变化最显著的4头个体（剪切力为19.740kg）和对照组（剪切力为5.040kg）之间背最长肌组织基因表达谱，发现137个差异表达基因（或EST）。其中64个在应激组中表达量上调，73个表达量下调。功能聚类分析和调控通路分析发现，这些基因涉及免疫反应和代谢调控等过程。初步确定免疫反应、脂肪代谢、分子转运、细胞之间信号传递和相互作用、细胞发育等通路和牛的肉质性状和应激状态相关。根据本次试验结果结合已有文献报道，初步确定interleukin12A (IL12A)、 interleukin13(IL13)、 chemokine (C-C motif) ligand8(CCL8)、chemokine (C-C motif) ligand24(CCL24)、 chemokine (C-X-C motif) ligand1(CXCL1)、activating transcription factor3(ATF3)、 regulator of Calcineurin1(RCAN1)、adiponectin、C1Q and collagen domain containing (ADIPOQ)、growth arrest and DNA-damage-inducible、 gamma (GADD45G)、 single-minded homolog1(SIM1)、 nuclearreceptor subfamily2， group F，member1(NR2F1)、zinc fingers and homeoboxes3(ZHX3)、GS homeobox2(GSX2)等基因可能是影响牛肉质性状和应激状况的重要基因。（3）利用miRNA芯片比较应激组和对照组背最长肌组织miRNA表达谱，发现bta-miR-497在应激组中的表达量显著升高。通过生物信息学的方法预测了bta-miR-497靶基因，其中70个基因为cDNA芯片中的差异表达基因。通过GO (Gene Ontology)Term和IPA (Ingiunty Pathway Analysis)通路分析，发现这70个基因主要参与到脂类代谢、脂肪酸合成、蛋白质磷酸化、神经系统发育等过程。从而推测bta-miR-497通过调控目标基因参与到上述通路中，进而影响牛肉的品质。（4）分析了HSPA1A等10个差异表达基因启动子区域DNA甲基化水平，发现应激组中HSPA1A启动子区域的DNA甲基化水平显著升高（P<0.05），而LOC614805启动子区域的DNA甲基化水平显著降低（P<0.05）。同时这2个基因的表达量也发生了显著变化，表达量变化和启动子区域DNA甲基化水平呈负相关。初步推断DNA甲基化通过调控基因的表达参与到肉质性状的调控中。（5）在不同嫩度的牛肉组中，利用Agilent基因芯片对其背最长肌组织样品进行差异表达基因分析，发现了53个基因（或EST）显著差异表达，其中27个基因（或EST）在嫩度差的牛肉中表达量上调，而26个基因（或EST）表达量下调。这些基因主要参与脂肪代谢等过程。初步确定脂肪代谢、胆固醇平衡、胆固醇运输、甾醇平衡、甾醇运输等通路和牛的肉质性状相关。根据前人的文献报道和本次研究结果，初步确定myosin, heavy chain3skeletal muscle embryonic (MYH3)、myosin heavy chain8skeletal muscle perinatal (MYH8)、 fatty acid binding protein4(FABP4)、 Stearoyl-coenzyme A desaturase (SCD)、Fatty acid synthase (FASN)、thyroid hormone responsive（THRSP）、ubiquitinlike with PHD and ring finger domains1(UHRF1)、guanylatebinding protein1(GBP5)、GBP3、GBP5、GBP6、GBP7等基因可能是影响牛肉质性状的重要基因。更多还原

【Abstract】 Although much research has been focused on beef tenderness, the mechanisms andfunctional genes of controlling beef quality have yet to be elucidated. In order to gain deepinsight into beef tenderness, in this research, we attempt to explore underlying genes,networks and pathways related to beef tenderness by evaluating the muscle transcriptomicprofiles in Angus cattle with divergent tenderness induced by acute stress. The results areshown below.(1) Beef quality varied significantly after acute stress. The Warner-Bratzler shear force(WBSF) of LD significantly increased after acute stress, and beef tenderness became worse.Fat acid contents of different stress status varied largely also. But the beef quality variationwithin stress group was divergent largely.(2) Agilent bovine4×44bovine microarray was used to analyze LD musles from3control and4stress individuals with highest WBSF. The results showed that137genessignificantly differentially expressed between stress and control groups （P<0.05，|logFC|>1.5，FDR<0.3）. Of which,64were up-regulated in stress group compared withcontrol group while73were down-regulated. Cluster analysis, Gene Ontology (Go) Term,and IPA (Ingenuity Pathway Analysis) results showed that these genes functioned ininflammatory response, lipid metabolism, molecular transport, cell-to-cell signaling andinteraction, cellular development, etc. Combine our results and literatures, we concluded thatgenes, such as IL12A, IL13, CCL8, CCL24, CXCL1, ATF3, RCAN1, ADIPOQ, GADD45G,SIM1, NR2F1, ZHX3, GSX2, may play roles in beef quality and anti-stress.（3）miRNA microarray was used to analyze the miRNA expression profiles between3control and3stress with highest WBSF. Microarray and qRT-PCR results showed that bta-miR-497was over-expressed in stress group compared to control group.1160genes werepredicted as this miRNA’s regulation targets using bioinformatic tools. Combined with Oligomicroarray, in these target genes,70were differentially expressed. These70genes wereinvolved in protein autophosphorylation, actin cytoskeleton organization, cerebral cortex cellmigration, actin filament-based process, retinoic acid receptor signaling pathway, endoplasmic reticulum organization, protein phosphorylation, etc. We concluded that miRNAinfluenced beef quality by regulating target genes and bta-miR-497may play some roles inbeef quality and stress-resistant.(4) DNA methylation level in promoters of differentially expressed genes significantlyvaried after acute stress. Gene expression was negatively associated with DNA metylationlevel. This confirmed that DNA methylation in promoter represses gene expression. Weinferred that DNA methylation may involve in beef quality varation in this experiment. Wealso found the change of DNA methylation in blood. This just gave us a clue that DNAmethylation lelvel in bovine blood may be a biomarker for beef quality and stress status.(5) Beef quality varied largely within stress group. Microarray was used to analyzetender and tough groups. We found53genes (EST) were significantly differentially expressed.Of which,27were up-regulated in tough group compared to tender group while26weredown-regulted. These genes were involved in lipid metabolism, etc. We concluded thathydrolase, peptidase, GTPase activity, lipid metabolism, small molecule biochemistry,molecular transport, and tissue development were related with beef quality variation. Weinferred that genes, such as FABP4、SCD、FASN、THRSP、MYH3、 MYH8、GBP1、GBP3、GBP5、GBP6、GBP7, could be candidate genes for beef quality and tenderness.更多还原