转录组测序解析氨气刺激后仔猪呼吸道和肝脏转录谱的变化

发布时间：2018-03-09 14:44

  本文选题：氨气　切入点：猪　出处：《华中农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：氨气是规模化猪场中普遍存在的有毒有害气体之一,较高浓度的氨气会给仔猪和工作人员带来健康隐患,而目前关于氨气影响仔猪健康的研究尚不足。本研究选取7周龄14 kg的保育仔猪共12头,随机分为4组,用56 mg/m3左右的氨气对3个试验组分别刺激4天、8天和12天,对照组则不作处理。刺激结束后采集仔猪肺,鼻黏膜和肝脏组织,提取总RNA,然后对这4个时期的样品进行转录组测序(RNA-seq),并探讨氨气刺激对3个组织转录谱的影响。主要结果:1.分别鉴定了3个组织氨气刺激前后的差异表达基因(默认和较小天数组比),肺和鼻黏膜一共6个比较(NC与3个处理组间的比较,3个处理组间的两两比较),肝脏只有1个比较(D12比NC)。统计显示:肺的差异表达基因一共有289个,鼻黏膜则有825个,肝脏有589个(367个上调表达,222个下调表达)。2.构建了肺和鼻黏膜差异表达基因的表达模式,并对每个模式的基因进行了功能富集分析。肺的差异表达基因一共被分为18类,其中SCGB3A2,MMPs,BPIFA1,IL8,ARG2,COMP,PAI-1,COLs,CLCA1,ELANE等与肺纤维化发生和气道黏液分泌相关的基因大部分处于cluster 1,cluster 2和cluster 3中。鼻黏膜的差异表达基因被分为了20类,其中cluster 1,cluster 2,cluster 3和cluster 6中CCDCs,CFAPs,TEKTs,DNAHs,LRRCs,SPAG17,COL11A2,ACAN,SNTN与黏膜功能、结构有关;cluster 4中SFTPs,BPIFBs与呼吸功能及防御反应相关;cluster 7中MASP1,LBP,C4BPA,IL1B,REG3G,S100As与急性炎症和黏液分泌有关;cluster 8和cluster 9的PAI-1,MMPs,KRTs还与上皮修复有关。3.肝脏差异表达基因显著富集的通路(p≤0.05)与免疫,癌症,肝病,氨基酸代谢及生理节奏有关。蛋白互作分析表明PIK3CA,TNF,NFKBIA,THBS1,JUN,HIF1A和FOS可能是重要的调控因子。4.鉴定了NC组和D12组间的差异可变剪切及差异表达转录本。结果显示CD40、SAT1、ETFB、CLK1、CHTOP和TNFRSF1A在鼻黏膜或者肺NC组和D12处理组间具有显著差异(FDR≤0.05)的可变剪接事件。同时,分别在肺、鼻黏膜和肝脏上发现了数量不等的差异表达(FDR≤0.05)的转录本。功能分析表明以上基因大部分与免疫,细胞凋亡和癌症有关。5.用22个样品的转录组测序数据鉴定了903条新lincRNA,并用权重基因共表达网络整合分析(Weighted gene co-expression network analysis,WGCNA)差异表达的linc基因和编码基因(共3072个)。结果显示LIPG在肝脏D12特异性高表达,TF在肝脏NC组特异性高表达;而新lincRNA XLOC_004910则在鼻黏膜D12特异性高表达,其临近的9个编码基因(上下游100 kb)则与免疫,糖/激素代谢,造血有关,暗示该lincRNA可能参与了这些进程。主要结论:氨气刺激引起仔猪鼻黏膜、肺和肝脏组织大量基因上调表达,其中刺激12天后差异表达基因数目最多。这些差异表达基因主要参与了呼吸道重塑、氧化应激、炎症和免疫应答等生物学进程。同时发现基因的选择性剪切和lincRNA在仔猪应对氨气刺激过程中也可能发挥重要的调控作用。
[Abstract]:Ammonia is one of the most common toxic and harmful gases in large-scale pig farms. A higher concentration of ammonia will bring health risks to piglets and workers. In this study, 12 piglets aged 7 weeks and 14 kg were randomly divided into 4 groups, which were stimulated with ammonia for about 56 mg/m3 for 4 days, 8 days and 12 days, respectively. The control group was not treated. The lungs, nasal mucosa and liver tissues of piglets were collected after stimulation. Total RNAs were extracted, and then transcriptome sequencing was carried out on the samples in these four periods. The effects of ammonia stimulation on three tissue transcriptional profiles were investigated. The main results were as follows: 1. The differentially expressed genes were identified before and after ammonia stimulation in three tissues. The default and smaller days of array comparison, lung and nasal mucosa a total of 6 comparison between the comparison between NC and 3 treatment groups, three treatment groups of pairwise comparison, liver only one comparison of D12 than NCC.statistics show: lung differentially expressed genes a total of 289, 825 nasal mucosa, 589 liver, 367 upregulated expression, 222 down-regulated expression genes were constructed to express differentially expressed genes in lung and nasal mucosa. The differentially expressed genes in the lung were divided into 18 types. Most of the genes related to pulmonary fibrosis and airway mucus secretion were found in cluster 1 cluster 2 and cluster 3. The differentially expressed genes in nasal mucosa were classified into 20 categories, including cluster 1 cluster 2 and cluster 6. Structurally related to cluster 4, SFTPslBPIFBs are associated with respiratory function and defense response in cluster 7. MASP1LBPU C4BPABPAIL1BNREG3GN S100As is associated with acute inflammation and mucus secretion. The PAI-1MMPsKRTs associated with epithelial repair are also associated with epithelial repair. The pathway of liver differentially expressed gene enrichment is less than 0.05) and immunity, cancer, cancer, and cancer. Liver disease, Amino acid metabolism and physiological rhythm are related. Protein interaction analysis shows that PIK3CAA TNFKBIATHBS1THBS1JUNHIF1A and FOS may be important regulatory factors. 4. The differential variable shear and differential expression transcripts between NC group and D12 group were identified. The results showed that CD40SAT1ETFBBK1 CHTOP and TNFRSF1A were present in nasal mucus. There was a significant difference between the membrane or lung NC group and the D12 treatment group in the variable splicing events (FDR 鈮，

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