草鱼抗菌免疫基因筛选及补体凝集素途径基因功能研究

发布时间：2018-03-12 08:07

  本文选题：草鱼　切入点：先天免疫　出处：《上海海洋大学》2017年博士论文　论文类型：学位论文

【摘要】：草鱼(Ctenopharyngodon idellus)是我国重要的淡水养殖鱼类之一。本论文利用高通量测序技术检测了草鱼感染嗜水气单胞菌(AH10)脾脏转录组。筛选草鱼抗菌免疫差异表达基因并研究了甘露糖结合凝集素(MBL)、甘露糖结合凝集素丝氨酸蛋白酶1(MASP-1)和补体C2的功能。研究内容及结果如下:1.草鱼脾脏mRNA转录组测序分析利用NextSeq 500技术平台,分别在7个时间点(0h、4h、8h、12h、24h、48h和72h)对腹腔注射AH10和PBS的草鱼脾脏cDNA文库进行高通量测序。获得149,514,670条raw reads,共42.67 G数据量。原始数据过滤后,共获得122,181,346条clean reads。经过拼接和组装后,191,795条基因注释到Nr、KO、GO、KOG、Swiss-Port和eggNOG数据库。通过筛选,获得2,992个免疫相关差异表达基因。参与到免疫系统的差异表达基因大多富集到补体系统中。基因差异表达分析和荧光定量PCR验证结果表明:(1)草鱼补体信号通路在抗菌免疫应答中起到重要的作用;(2)补体系统在草鱼感染4h后被激活;(3)补体基因mRNA呈波动表达模式,表明草鱼补体系统可能有信号传递的功能。2.MBL在抗菌感染中介导的免疫应答MBL能激活补体凝集素途径,在哺乳动物和硬骨鱼类中均起到重要的免疫作用。草鱼MBL(gcMBL)的cDNA全长为984 bp,包含741 bp的开放阅读框(ORF),编码246个氨基酸。gcMBL广泛分布在草鱼各组织中,表达量能被AH10、脂多糖(LPS)和鞭毛蛋白(FLA)影响。过表达细胞中发现除C5外,凝集素途径的下游分子在转录水平上均显著上调。用AH10、LPS和FLA分别刺激过表达细胞,发现补体受体、补体调节蛋白和炎症相关因子的表达量均显著上调。用AH10侵染敲减gcMBL的肝脏原代细胞,与对照组比较发现,上述各因子的表达量均被抑制。利用双荧光素酶检测系统研究发现,过表达gcMBL能显著激活NF-κB信号。综上所述,gcMBL基因在草鱼抗细菌侵染中起到重要的免疫作用。3.草鱼甘露糖结合凝集素丝氨酸蛋白酶1的免疫功能的研究露糖结合凝集素丝氨酸蛋白酶(MASPs)是补体系统凝集素途径的重要组分,本研究利用转录组数据库中得到的草鱼MASP-1(gcMASP-1)序列进行研究,发现gcMASP-1基因的cDNA全长3308 bp,ORF全长为2160 bp,编码719个氨基酸,gcMASP-1在各物种间保守程度较高。组织表达谱分析结果显示,gcMASP-1基因广泛分布在各个组织中,相对较高的在心脏、肝脏和脑组织中表达。利用AH10侵染草鱼,发现gcMASP-1的表达量呈波动性变化;利用AH10、LPS和FLA刺激草鱼肝脏原代细胞,结果表明gcMASP-1的表达量随刺激时间的变化呈波动变化。在草鱼肝细胞中过表达gcMASP-1后,凝集素通路上游基因的表达量没有显著变化;但下游基因除C5外的表达水平均显著提高;同时,也可以诱导免疫相关基因gcil-1β,gcIFN和gcTNF-α的表达。利用AH10刺激过表达或抑制表达gcMASP-1的细胞,结果表明,过表达细胞中炎症因子等的表达量均显著上调;在敲减细胞中,上述因子等表达量被显著抑制。综上所述,gcMASP-1在草鱼凝集素途径中起到关键的抗菌免疫功能。4.草鱼补体C2在抗菌侵疫功中的作用和功能补体C2是凝集素途径中形成C3转化酶的重要组分。草鱼C2(gcC2)的cDNA全长为2684 bp,包含2523 bp的ORF,编码840个氨基酸,蛋白序列在物种间保守。gcC2的mRNA在草鱼各组织中均有表达。AH10能在草鱼体内和体外影响gcC2的表达量。同时,LPS和FLA能引起肝脏原代细胞中gcC2表达量的改变。在肝脏原代细胞中过表达gcC2能引起除C5外的补体系统下游组分表达量升高。利用AH10、LPS或FLA刺激过表达或敲减gcC2的肝脏细胞,结果显示,在过表达细胞中炎症因子等的表达量显著提高;在敲减细胞中被显著抑制。双荧光报告检测系统结果表明gcC2基因能激活核转录因子应答AH10感染。综上所述,gcC2基因在草鱼的先天免疫和激活补体下游组分中起到重要作用。
[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，

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