β-葡聚糖诱导斜带石斑鱼转录组分析及免疫相关基因表达研究

发布时间：2018-03-02 19:27

  本文选题：斜带石斑鱼　切入点：β-葡聚糖　出处：《广东海洋大学》2017年硕士论文　论文类型：学位论文

【摘要】：斜带石斑鱼(Epinephelus coioides)是我国重要的海水经济养殖鱼类之一,然而其养殖产量一直受到病害的制约。β-葡聚糖作为一种新型的天然免疫增强剂,在水产养殖业中具有良好的应用前景。本文开展了β-葡聚糖诱导斜带石斑鱼转录组测序分析及不同浓度β-葡聚糖诱导下免疫相关基因的表达分析,探究了β-葡聚糖对斜带石斑鱼免疫相关基因表达的影响。通过腹腔注射0.2 m Lβ-葡聚糖(浓度0.05 g/m L)刺激斜带石斑鱼,分别于6 h、72 h分别取胸腺、脾脏、头肾和血液淋巴细胞四个组织进行转录组测序。结果一共组装了123677条unigene,平均长度是1711.30 bp。通过Blast比对数据库进行功能注释,一共注释了96355条unigene。六大数据库注释得到的unigene数量分别为:91955条(Nt)、66089条(Nr)、14126条(GO)、24018条(COG)、9673条(KEGG)和54148条(Swiss Prot)。数据结果表明组装效果良好,据此进行转录组分析得出:(1)对差异表达基因进行分析,筛选出6类免疫相关的显著性差异基因74个,涉及抗原加工呈递、细胞粘附分子、补体系统、细胞因子、模式识别受体和吞噬作用。(2)对差异基因进行GO富集分析,筛选出免疫相关条目7条,包括免疫应答、B细胞受体信号通路、趋化因子受体活性、白细胞趋化性、趋化因子调节信号通路、白细胞介素-1受体绑定和溶酶体组份。(3)对差异基因进行KEGG通路富集分析,共筛选出19类免疫相关通路。其中Tolllike受体信号通路和RIG-I-like受体信号通路在血液淋巴细胞、脾脏、头肾和胸腺四个免疫组织均显著富集;T细胞受体信号通路、NOD-like受体信号通路、自然杀伤细胞介导的细胞毒性通路、造血细胞谱系通路在血液淋巴细胞、脾脏和头肾三个组织中均显著富集;B细胞受体信号通路和肠道Ig产生的免疫网络通路在血液淋巴细胞和头肾组织中均显著富集。(4)随机挑选7个显著性差异表达的免疫相关基因进行q RT-PCR验证,验证结果与测序的变化趋势一致,说明转录组测序结果的可靠性较高。进一步研究在不同浓度的β-葡聚糖诱导下,斜带石斑鱼免疫相关基因时序表达情况。实验设置5个实验组,分别对斜带石斑鱼注射0.2 m L的b-葡聚糖(浓度为0.015 g/m L、0.03 g/m L、0.06g/m L、0.12g/m L、0.15 g/m L),1个对照组注射等量的PBS缓冲液。在注射后的1d、2d、3d、4d、5d、6d和7d分别采集胸腺、脾脏、头肾和血液淋巴细胞四个组织样本。最后通过Real-Time PCR技术检测斜带石斑鱼的IL-1β、TLR13、IFNα、MHC-I、TNFα、C3-1和CD2基因的相对表达量。结果显示:(1)IL-1β、TNFα和TLR13基因的相对表达量受到β-葡聚糖注射剂量的影响:三者都是在注射浓度为0.06 g/m L时,基因整体表达量达到峰值;浓度低于0.06g/m L时,表达量随着浓度的升高而升高;当浓度高于0.12g/m L时,基因的表达量随着浓度升高而下降。(2)MHC-I基因和C3-1基因的表达量受到β-葡聚糖注射剂量的影响:注射浓度为0.06 g/m L时,二者整体相对表达量达到峰值;浓度大于0.12 g/m L时,基因的表达量一直维持在相对较高水平,但并不随β-葡聚糖浓度的增加而升高。(3)IFNα基因表达量受到β-葡聚糖注射剂量的影响:注射浓度为0.12g/m L时,四个组织中的相对表达量达到最高;浓度低于0.12 g/m L时其表达量随着浓度的升高而升高。(4)CD2基因的表达与β-葡聚糖刺激的浓度和刺激后的时间都有一定的关系:当浓度大于0.12g/m L时,基因的相对表达量从5d以后显著提高。本论文研究工作为β-葡聚糖调节鱼类免疫系统的作用机制提供参考。
[Abstract]:Grouper (Epinephelus coioides) is one of China's economically important marine fish species, but its aquaculture production has been restricted. The diseases of beta glucan as a new natural immune enhancer, it has a good application prospect in the aquaculture industry. This paper carried out the analysis of the expression of beta glucan induced e.coioides fish transcriptome sequencing and different concentrations of beta glucan induced genes related to immune, explores the effects of beta glucan on grouper immune related genes expression. By intraperitoneal injection of 0.2 m L (beta glucan concentration 0.05 g/m L) stimulate grouper, respectively 6 h and 72 h respectively. Thymus, spleen, head kidney and peripheral blood lymphocytes of four tissue transcriptome sequencing. Results a total of 123677 UniGene were assembled, the average length of 1711.30 bp. were annotated by Blast comparison database, a note Release the number of UniGene 96355 unigene. six database annotated from respectively: 91955 (Nt), 66089 (Nr), 14126 (GO), 24018 (COG), 9673 (KEGG) and 54148 (Swiss Prot). The data showed that the assembly of good effect, accordingly. Group analysis: (1) we analyzed the differentially expressed genes, screened genes significantly related to 6 types of immune 74, involved in antigen processing and presentation, cell adhesion molecules, cytokines, complement system, pattern recognition receptors and phagocytosis. (2) GO enrichment analysis of differentially expressed genes, screened out by the some 7 items, including immune responses, B cell receptor signaling pathway, chemokine receptor activity, leukocyte chemotaxis, chemokine signaling pathways regulating, interleukin -1 receptor binding and lysosomal components. (3) the difference for KEGG pathway enrichment analysis, we identified 19 types of immune related Which pathway. Tolllike receptor signaling pathway and RIG-I-like receptor signaling pathway in blood lymphocytes, spleen, head kidney and thymus of four immune tissues were significantly enriched; T cell receptor signaling pathway, NOD-like signaling pathway, natural killer cell cytotoxicity mediated pathway, hematopoietic cell lineage pathway in blood lymphocytes, spleen and head kidney three tissues were enriched; immune network pathway of B cell receptor signaling pathway and intestinal Ig production in blood lymphocytes and head kidney tissues were significantly enriched. (4) Q RT-PCR verified the immune related genes of randomly selected 7 significant differential expression, consistent results and sequencing, reliability the higher transcriptome sequencing results. Further research in different concentrations of beta glucan induced by grouper gene expression sequence. The experimental set 5 涓�瀹為獙缁�,鍒嗗埆瀵规枩甯︾煶鏂戦奔娉ㄥ皠0.2 m L鐨刡-钁¤仛绯，

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