本文選題：草魚　切入點(diǎn)：先天免疫　出處：《上海海洋大學(xué)》2017年博士論文　論文類型：學(xué)位論文

【摘要】：草魚(Ctenopharyngodon idellus)是我國(guó)重要的淡水養(yǎng)殖魚類之一。本論文利用高通量測(cè)序技術(shù)檢測(cè)了草魚感染嗜水氣單胞菌(AH10)脾臟轉(zhuǎn)錄組。篩選草魚抗菌免疫差異表達(dá)基因并研究了甘露糖結(jié)合凝集素(MBL)、甘露糖結(jié)合凝集素絲氨酸蛋白酶1(MASP-1)和補(bǔ)體C2的功能。研究?jī)?nèi)容及結(jié)果如下:1.草魚脾臟mRNA轉(zhuǎn)錄組測(cè)序分析利用NextSeq 500技術(shù)平臺(tái),分別在7個(gè)時(shí)間點(diǎn)(0h、4h、8h、12h、24h、48h和72h)對(duì)腹腔注射AH10和PBS的草魚脾臟cDNA文庫(kù)進(jìn)行高通量測(cè)序。獲得149,514,670條raw reads,共42.67 G數(shù)據(jù)量。原始數(shù)據(jù)過(guò)濾后,共獲得122,181,346條clean reads。經(jīng)過(guò)拼接和組裝后,191,795條基因注釋到Nr、KO、GO、KOG、Swiss-Port和eggNOG數(shù)據(jù)庫(kù)。通過(guò)篩選,獲得2,992個(gè)免疫相關(guān)差異表達(dá)基因。參與到免疫系統(tǒng)的差異表達(dá)基因大多富集到補(bǔ)體系統(tǒng)中�；虿町惐磉_(dá)分析和熒光定量PCR驗(yàn)證結(jié)果表明:(1)草魚補(bǔ)體信號(hào)通路在抗菌免疫應(yīng)答中起到重要的作用;(2)補(bǔ)體系統(tǒng)在草魚感染4h后被激活;(3)補(bǔ)體基因mRNA呈波動(dòng)表達(dá)模式,表明草魚補(bǔ)體系統(tǒng)可能有信號(hào)傳遞的功能。2.MBL在抗菌感染中介導(dǎo)的免疫應(yīng)答MBL能激活補(bǔ)體凝集素途徑,在哺乳動(dòng)物和硬骨魚類中均起到重要的免疫作用。草魚MBL(gcMBL)的cDNA全長(zhǎng)為984 bp,包含741 bp的開(kāi)放閱讀框(ORF),編碼246個(gè)氨基酸。gcMBL廣泛分布在草魚各組織中,表達(dá)量能被AH10、脂多糖(LPS)和鞭毛蛋白(FLA)影響。過(guò)表達(dá)細(xì)胞中發(fā)現(xiàn)除C5外,凝集素途徑的下游分子在轉(zhuǎn)錄水平上均顯著上調(diào)。用AH10、LPS和FLA分別刺激過(guò)表達(dá)細(xì)胞,發(fā)現(xiàn)補(bǔ)體受體、補(bǔ)體調(diào)節(jié)蛋白和炎癥相關(guān)因子的表達(dá)量均顯著上調(diào)。用AH10侵染敲減gcMBL的肝臟原代細(xì)胞,與對(duì)照組比較發(fā)現(xiàn),上述各因子的表達(dá)量均被抑制。利用雙熒光素酶檢測(cè)系統(tǒng)研究發(fā)現(xiàn),過(guò)表達(dá)gcMBL能顯著激活NF-κB信號(hào)。綜上所述,gcMBL基因在草魚抗細(xì)菌侵染中起到重要的免疫作用。3.草魚甘露糖結(jié)合凝集素絲氨酸蛋白酶1的免疫功能的研究露糖結(jié)合凝集素絲氨酸蛋白酶(MASPs)是補(bǔ)體系統(tǒng)凝集素途徑的重要組分,本研究利用轉(zhuǎn)錄組數(shù)據(jù)庫(kù)中得到的草魚MASP-1(gcMASP-1)序列進(jìn)行研究,發(fā)現(xiàn)gcMASP-1基因的cDNA全長(zhǎng)3308 bp,ORF全長(zhǎng)為2160 bp,編碼719個(gè)氨基酸,gcMASP-1在各物種間保守程度較高。組織表達(dá)譜分析結(jié)果顯示,gcMASP-1基因廣泛分布在各個(gè)組織中,相對(duì)較高的在心臟、肝臟和腦組織中表達(dá)。利用AH10侵染草魚,發(fā)現(xiàn)gcMASP-1的表達(dá)量呈波動(dòng)性變化;利用AH10、LPS和FLA刺激草魚肝臟原代細(xì)胞,結(jié)果表明gcMASP-1的表達(dá)量隨刺激時(shí)間的變化呈波動(dòng)變化。在草魚肝細(xì)胞中過(guò)表達(dá)gcMASP-1后,凝集素通路上游基因的表達(dá)量沒(méi)有顯著變化;但下游基因除C5外的表達(dá)水平均顯著提高;同時(shí),也可以誘導(dǎo)免疫相關(guān)基因gcil-1β,gcIFN和gcTNF-α的表達(dá)。利用AH10刺激過(guò)表達(dá)或抑制表達(dá)gcMASP-1的細(xì)胞,結(jié)果表明,過(guò)表達(dá)細(xì)胞中炎癥因子等的表達(dá)量均顯著上調(diào);在敲減細(xì)胞中,上述因子等表達(dá)量被顯著抑制。綜上所述,gcMASP-1在草魚凝集素途徑中起到關(guān)鍵的抗菌免疫功能。4.草魚補(bǔ)體C2在抗菌侵疫功中的作用和功能補(bǔ)體C2是凝集素途徑中形成C3轉(zhuǎn)化酶的重要組分。草魚C2(gcC2)的cDNA全長(zhǎng)為2684 bp,包含2523 bp的ORF,編碼840個(gè)氨基酸,蛋白序列在物種間保守。gcC2的mRNA在草魚各組織中均有表達(dá)。AH10能在草魚體內(nèi)和體外影響gcC2的表達(dá)量。同時(shí),LPS和FLA能引起肝臟原代細(xì)胞中g(shù)cC2表達(dá)量的改變。在肝臟原代細(xì)胞中過(guò)表達(dá)gcC2能引起除C5外的補(bǔ)體系統(tǒng)下游組分表達(dá)量升高。利用AH10、LPS或FLA刺激過(guò)表達(dá)或敲減gcC2的肝臟細(xì)胞,結(jié)果顯示,在過(guò)表達(dá)細(xì)胞中炎癥因子等的表達(dá)量顯著提高;在敲減細(xì)胞中被顯著抑制。雙熒光報(bào)告檢測(cè)系統(tǒng)結(jié)果表明gcC2基因能激活核轉(zhuǎn)錄因子應(yīng)答AH10感染。綜上所述,gcC2基因在草魚的先天免疫和激活補(bǔ)體下游組分中起到重要作用。
[Abstract]:Grass carp (Ctenopharyngodon idellus) is one of China's important freshwater fishes. This paper use high-throughput sequencing to detect grass carp infection of Aeromonas hydrophila (AH10) spleen transcriptome. Screening of differentially expressed genes grass carp antibacterial immunity and study of mannose binding lectin (MBL), mannose binding lectin serine protease 1 (MASP-1) and complement the function of C2. The research contents and results are as follows: 1. grass carp spleen mRNA transcriptome sequencing analysis using NextSeq 500 platform, respectively at 7 time points (0h, 4h, 8h, 12h, 24h, 48h and 72h) for intraperitoneal injection of AH10 and PBS grass carp spleen cDNA library by high-throughput sequencing. 149514670 raw reads, a total of 42.67 G data. The original data after filtering, received a total of 122181346 clean reads. after assembled and after 191795 gene annotation to Nr, KO, GO, KOG, Swiss-Port and eggNOG database. After screening, gene expression of 2992 immune related differences. Differences involved in the immune system genes are enriched to the complement system. Expression analysis and fluorescence quantitative PCR results showed that the differentially expressed genes: (1) grass carp complement pathway plays an important role in the antibacterial immune response; (2) the complement system is activated in the grass carp 4h after infection; (3) mRNA fluctuating complement gene expression patterns showed that the complement system may have the function of grass carp.2.MBL signaling in the antibacterial immune response to infection MBL mediated activation of the lectin complement pathway, in mammals and teleosts play an important immune function. The grass carp MBL (gcMBL) cDNA the total length of 984 BP, contains 741 BP open reading frame (ORF), encoding 246 amino acid.GcMBL is widely distributed in various tissues can be expressed in grass carp, AH10, lipopolysaccharide (LPS) and flagellin (FLA). Over the table Was found in cells in addition to C5, the downstream molecule of the lectin pathway at the transcriptional level were significantly up-regulated. AH10, LPS and FLA were used to stimulate expression of cells, found that complement receptor, expression of complement regulatory proteins and inflammatory factors were significantly up-regulated. The primary infection by AH10 knockdown of gcMBL liver cells, comparison found with the control group, the expression of the factors were suppressed. Using dual luciferase detection system research found that overexpression of gcMBL could activate NF- kappa B signal. To sum up, the research of lectin binding serine protease 1 immune function plays an important role in the immune mannose.3. grass carp gcMBL gene in grass carp anti bacterial infection in the dew sugar binding lectin serine protease (MASPs) is an important complement of the lectin pathway, this study use grass carp MASP-1 to get the transcriptome database (gcMASP-1) sequence. We found that the gcMASP-1 gene of cDNA was 3308 BP, ORF was 2160 BP, encoding 719 amino acids in each gcMASP-1 are conserved between species of high degree. The spectrum analysis result showed that the expression of gcMASP-1 gene, is widely distributed in various tissues, relatively high expression in the heart, liver and brain tissues. AH10 infection by grass carp the expression of gcMASP-1, found a fluctuation; using AH10, LPS and FLA stimulate grass carp liver primary cells, results showed that the expression of gcMASP-1 changes with the stimulation time fluctuated. Overexpression of gcMASP-1 in grass carp liver cells after the expression of lectin pathway upstream genes did not change significantly; but the expression level of downstream genes except C5 were significantly increased; at the same time, can also induce immune related gene expression of gcIFN and gcil-1 beta, gcTNF- alpha. Stimulated by AH10 overexpression or inhibition of the expression of gcMASP-1 cells, results table 鏄，

本文編號(hào)：1600693