本文選題：斜帶石斑魚　切入點：β-葡聚糖　出處：《廣東海洋大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：斜帶石斑魚(Epinephelus coioides)是我國重要的海水經(jīng)濟養(yǎng)殖魚類之一,然而其養(yǎng)殖產(chǎn)量一直受到病害的制約。β-葡聚糖作為一種新型的天然免疫增強劑,在水產(chǎn)養(yǎng)殖業(yè)中具有良好的應(yīng)用前景。本文開展了β-葡聚糖誘導(dǎo)斜帶石斑魚轉(zhuǎn)錄組測序分析及不同濃度β-葡聚糖誘導(dǎo)下免疫相關(guān)基因的表達分析,探究了β-葡聚糖對斜帶石斑魚免疫相關(guān)基因表達的影響。通過腹腔注射0.2 m Lβ-葡聚糖(濃度0.05 g/m L)刺激斜帶石斑魚,分別于6 h、72 h分別取胸腺、脾臟、頭腎和血液淋巴細(xì)胞四個組織進行轉(zhuǎn)錄組測序。結(jié)果一共組裝了123677條unigene,平均長度是1711.30 bp。通過Blast比對數(shù)據(jù)庫進行功能注釋,一共注釋了96355條unigene。六大數(shù)據(jù)庫注釋得到的unigene數(shù)量分別為:91955條(Nt)、66089條(Nr)、14126條(GO)、24018條(COG)、9673條(KEGG)和54148條(Swiss Prot)。數(shù)據(jù)結(jié)果表明組裝效果良好,據(jù)此進行轉(zhuǎn)錄組分析得出:(1)對差異表達基因進行分析,篩選出6類免疫相關(guān)的顯著性差異基因74個,涉及抗原加工呈遞、細(xì)胞粘附分子、補體系統(tǒng)、細(xì)胞因子、模式識別受體和吞噬作用。(2)對差異基因進行GO富集分析,篩選出免疫相關(guān)條目7條,包括免疫應(yīng)答、B細(xì)胞受體信號通路、趨化因子受體活性、白細(xì)胞趨化性、趨化因子調(diào)節(jié)信號通路、白細(xì)胞介素-1受體綁定和溶酶體組份。(3)對差異基因進行KEGG通路富集分析,共篩選出19類免疫相關(guān)通路。其中Tolllike受體信號通路和RIG-I-like受體信號通路在血液淋巴細(xì)胞、脾臟、頭腎和胸腺四個免疫組織均顯著富集;T細(xì)胞受體信號通路、NOD-like受體信號通路、自然殺傷細(xì)胞介導(dǎo)的細(xì)胞毒性通路、造血細(xì)胞譜系通路在血液淋巴細(xì)胞、脾臟和頭腎三個組織中均顯著富集;B細(xì)胞受體信號通路和腸道Ig產(chǎn)生的免疫網(wǎng)絡(luò)通路在血液淋巴細(xì)胞和頭腎組織中均顯著富集。(4)隨機挑選7個顯著性差異表達的免疫相關(guān)基因進行q RT-PCR驗證,驗證結(jié)果與測序的變化趨勢一致,說明轉(zhuǎn)錄組測序結(jié)果的可靠性較高。進一步研究在不同濃度的β-葡聚糖誘導(dǎo)下,斜帶石斑魚免疫相關(guān)基因時序表達情況。實驗設(shè)置5個實驗組,分別對斜帶石斑魚注射0.2 m L的b-葡聚糖(濃度為0.015 g/m L、0.03 g/m L、0.06g/m L、0.12g/m L、0.15 g/m L),1個對照組注射等量的PBS緩沖液。在注射后的1d、2d、3d、4d、5d、6d和7d分別采集胸腺、脾臟、頭腎和血液淋巴細(xì)胞四個組織樣本。最后通過Real-Time PCR技術(shù)檢測斜帶石斑魚的IL-1β、TLR13、IFNα、MHC-I、TNFα、C3-1和CD2基因的相對表達量。結(jié)果顯示:(1)IL-1β、TNFα和TLR13基因的相對表達量受到β-葡聚糖注射劑量的影響:三者都是在注射濃度為0.06 g/m L時,基因整體表達量達到峰值;濃度低于0.06g/m L時,表達量隨著濃度的升高而升高;當(dāng)濃度高于0.12g/m L時,基因的表達量隨著濃度升高而下降。(2)MHC-I基因和C3-1基因的表達量受到β-葡聚糖注射劑量的影響:注射濃度為0.06 g/m L時,二者整體相對表達量達到峰值;濃度大于0.12 g/m L時,基因的表達量一直維持在相對較高水平,但并不隨β-葡聚糖濃度的增加而升高。(3)IFNα基因表達量受到β-葡聚糖注射劑量的影響:注射濃度為0.12g/m L時,四個組織中的相對表達量達到最高;濃度低于0.12 g/m L時其表達量隨著濃度的升高而升高。(4)CD2基因的表達與β-葡聚糖刺激的濃度和刺激后的時間都有一定的關(guān)系:當(dāng)濃度大于0.12g/m L時,基因的相對表達量從5d以后顯著提高。本論文研究工作為β-葡聚糖調(diào)節(jié)魚類免疫系統(tǒng)的作用機制提供參考。
[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疄楠岀粍,鍒嗗埆瀵規(guī)枩甯︾煶鏂戦奔娉ㄥ皠0.2 m L鐨刡-钁¤仛緋，

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