免疫细胞因子基因及相关miRNAs在肉鸡急性冷、热应激中的表达调控研究

发布时间：2018-01-22 22:03

本文关键词： 肉鸡急性冷/热应激实时荧光定量 miRNA 双荧光素酶　出处：《西北农林科技大学》2017年硕士论文　论文类型：学位论文

【摘要】：高密度集约化的饲养模式极易使肉鸡产生热应激。但是,湿帘以及降温措施不当使用又极易产生冷应激。急性冷、热应激的发生首先破坏的是家禽机体的免疫机能,严重影响肉鸡的商品化生产,造成巨大的经济损失。采用现代生物检测技术研究急性冷热应激条件下肉鸡免疫细胞因子基因在各免疫器官中的表达规律,揭示micRNAs与其靶基因对急性应激的调控机理,为培育抗寒抗热肉鸡品种提供理论依据,具有重大的实际生产意义。本实验以45只28日龄体重及健康状况相近的罗斯308肉鸡为研究对象,分为正常对照组、热应激组和冷应激组分别模拟实际生产产生急性热应激反应和冷应激反应。采用实时荧光定量PCR对8个免疫细胞因子基因(IL-1、IL-1α、IL-2、IL-4、IL-6、IL-12α、IL-12β、INF-γ)和4个热应激蛋白基因(Hsp60、Hsp70、Hsp90和Hsp110)在脾脏、胸腺、法氏囊、肝脏、心脏等组织中的表达量进行测定分析。并采用miRNA测序技术对脾脏组织的miRNAs进行检测,筛选差异表达miRNAs,并预测其靶基因。进一步从细胞水平和分子水平对差异表达mi RNAs与细胞因子基因互作进行验证,揭示差异miRNAs及其靶基因对急性冷热应激的调控机理。研究得到以下结果:1.在脾脏组织中,只有急性热应激条件下IL-1α和IL-1两个基因表达影响显著升高(P0.05);在胸腺组织中,除IL-12α表达在各处理组无显著变化,其余7个细胞因子表达量均显著下降;在法氏囊组织中,各处理组均未检测到IL-12α的表达,急性热应激条件下其他7个细胞因子基因表达均显著下降(P0.05)。2.在心脏组织与肝脏组织中,急性热应激条件下HSP70与HSP110表达极显著升高(P0.01)。3.对脾脏组织mi RNA测序结果揭示,根据|log2Fold|1和p value0.05原则,正常组和热应激组之间筛选出12个差异表达miRNAs,在正常组和冷应激组之间发现1个差异表达miRNAs,在冷应激和热应激组之间筛选出20个差异表达miRNAs。通过对所有差异表达miRNAs把基因的预测,筛选出4个差异表达mi RNAs的靶基因为免疫细胞因子基因。4.通过WEGO和GO Pathway通路富集分析,急性热应激条件下差异表达miRNAs显著富集在细胞因子互作的通路中。5.双荧光素酶报告结果表明,gga-miR-34a-5P和gga-miR-449c-5P对IL-2和IL-12α的表达具有负调控的作用。综上所述,本研究结果表明,急性热应激对免疫细胞因子的表达影响较大,尤其对胸腺组织中免疫细胞因子基因表达影响最大,而冷应激影响较小;各免疫细胞因子在不同组织中表达规律不一致;急性应激条件下,热应激蛋白Hsp70和Hsp110的表达极显著上调(P0.01)。gga-mi R-34a-5p和gga-miR-449c-5p对细胞因子基因IL-2与IL-12有极显著下调作用(P0.01),抑制了IL-2与IL-12表达水平。
[Abstract]:The high density intensive feeding mode can easily cause heat stress in broilers. However, cold stress is easy to be produced when wet curtains and cooling measures are not used properly. The occurrence of heat stress is the first damage to the immune function of poultry, seriously affecting the commercial production of broilers. Modern biological detection technique was used to study the expression of immune cytokine gene in immune organs of broilers under acute cold and heat stress. To reveal the regulation mechanism of micRNAs and its target gene on acute stress and to provide theoretical basis for breeding cold-resistant and heat-resistant broiler breeds. In this study, 45 rose-308 broilers with similar body weight and health status were divided into normal control group. The heat stress group and the cold stress group simulated the actual production to produce acute heat stress response and cold stress response respectively. IL-2, IL-4, IL-6, IL-12 伪, IL-12 尾, INF- 纬) and four heat stress protein genes, Hsp60, Hsp70. Hsp90 and Hsp110) in spleen, thymus, bursa of Fabricius, liver. The expression of miRNAs in spleen tissue was detected by miRNA sequencing, and the differential expression of miRNAs was screened. The target genes were predicted and the interaction between the differentially expressed mi RNAs and cytokine genes was further verified at the cellular and molecular levels. To reveal the regulation mechanism of differential miRNAs and its target gene on acute cold and heat stress. The following results were obtained: 1. In spleen tissue. Under acute heat stress, the expression of IL-1 伪 and IL-1 genes increased significantly (P 0.05). In thymus tissues, the expression of IL-12 伪 did not change significantly in all treatment groups, but the expression of other seven cytokines decreased significantly. No expression of IL-12 伪 was detected in the tissues of bursa of Fabricius. The expression of other seven cytokines genes decreased significantly under acute heat stress in heart and liver tissues. The expression of HSP70 and HSP110 was significantly increased under acute heat stress. The results of mi RNA sequencing in spleen tissue revealed that the expression of Mi RNA was significantly higher than that of control group (P 0.01). According to the principle of log2Fold 1 and p value0.05, 12 differentially expressed miRNAs were screened between normal group and heat stress group. There was a difference in expression of miRNAs between normal group and cold stress group. Twenty differentially expressed miRNAs were screened between cold stress and heat stress groups. All differentially expressed miRNAs genes were predicted. Four differentially expressed targets of mi RNAs were screened because of the immunocyte factor gene. 4. Enrichment analysis of WEGO and go Pathway pathway was carried out. The differential expression of miRNAs was significantly enriched in the cytokine interaction pathway under acute heat stress. Gga-miR-34a-5P and gga-miR-449c-5P play a negative role in the expression of IL-2 and IL-12 伪. Acute heat stress had a great effect on the expression of immune cytokines, especially on the gene expression of immune cytokines in thymus tissues, but cold stress had little effect on the expression of immune cytokines. The expression of immune cytokines in different tissues was not consistent. Under acute stress. The expression of heat stress protein (Hsp70) and heat stress protein (Hsp110) were significantly up-regulated (P0.01). GGa-mi R-34a-5p and gga-miR-449c-5p significantly down-regulated cytokine gene IL-2 and IL-12 (P0.01). The expression of IL-2 and IL-12 was inhibited.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.31

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