【作者】 黄腾；

【导师】 苏建国；

【作者基本信息】 西北农林科技大学 ， 水生生物学， 2010， 硕士

【摘要】 草鱼(Ctenopharyngodon idella)是我国重要的淡水经济鱼类,养殖规模居“四大家鱼”之首。在具有生长快、肉质好、养殖成本低等优点的同时,草鱼对多种病害的抵御能力较弱是养殖过程中面临的棘手问题之一,每年因病害造成的损失高达数十亿元。由草鱼呼肠孤病毒(Grass carp reovirus, GCRV)引起的草鱼出血病是主要病害之一。目前尚无特别有效的药物和方法针对该病毒,而草鱼自身特异性免疫能力不发达在某种程度上导致了疫苗使用效果不佳,因而从先天性免疫途径入手寻找抗GCRV的关键分子为今后草鱼抗病育种奠定基础。在漫长的物种进化过程中多细胞动物保留了许多相似的抗病毒先天性免疫途径,其中以TLR(Toll-like receptors)和RLR(RIG-I-like receptors)等模式识别家族途径为代表。RLR家族途径是哺乳动物中新近发现的重要的抗病毒途径之一,该家族包括3个成员:RIG-I(Retinoic acid-inducible gene-I)、MDA5(Melanoma-differentiation-associated gene 5)和LGP2(Laboratory of genetics and physiology 2),它们各自识别不同类型的胞内病毒核酸分子,并最终激发宿主产生一系列的抗病毒反应,因此阐明鱼类RLR家族在抗病毒过程中的作用具有重要意义。本研究根据基因序列同源比对的结果设计简并引物扩增部分片段,结合cDNA末端快速扩增、半定量RT-PCR、实时荧光定量RT-PCR及生物信息学分析等技术,克隆了草鱼MDA5 (CiMDA5)和LGP2(CiLGP2)基因,并就其进化关系以及在抗GCRV中发挥的作用进行了相关探索,取得了如下结果:(1) CiMDA5基因的克隆与表达胞浆解旋酶MDA5(黑色素瘤分化相关基因5)识别长的双链RNA病毒分子和5’磷酸基团的单链RNA,并介导I型干扰素的分泌。本研究首次从鱼类中克隆到MDA5基因,并通过研究CiMDA5基因的表达模式揭示其功能。CiMDA5基因的cDNA全长由3233个核苷酸构成,编码含961个氨基酸的蛋白。预测的氨基酸序列主要包括6个结构域:1个CARD(Caspase activation and recruitment domain)、1个DEXDc(DEAD/DEAH box helicase domain)、1个ResIII(Conserved restriction domain of bacterial type III restriction enzyme)、2个HELICc(Helicase superfamily c-terminal domain)和1个RD(Regulatory domain)调控区。CiMDA5基因的mRNA在所有被检组织中均有表达。半定量RT-PCR结果表明CiMDA5基因在正常个体的脾脏、皮肤和鳃的表达量较高,注射GCRV后表达量上调。脾脏中12 h的表达量显著上升(1.80倍,P<0.05),24 h达到峰值(7.48倍,P<0.05),注射后48 h恢复到正常水平(P>0.05)。肝脏中48 h的表达量显著上升(5.00倍,P<0.05),注射后72 h恢复到正常水平(P>0.05)。(2) CiLGP2基因的克隆与表达LGP2与RIG-I和MDA5同源,但缺少CARD结构域。本研究从草鱼中克隆了LGP2基因并对其表达规律进行了研究。CiLGP2基因的cDNA有2920个核苷酸,含5个ATTTA不稳定模体。CiLGP2基因的开放阅读框有2043个核苷酸,编码含680个氨基酸的蛋白,该预测蛋白包括5个主要重叠结构域:2个DEXDc、1个ResIII、1个HELICc以及1个RD调控区。通过同源建模,发现CiLGP2蛋白的RD调控区比人类的LGP2多一个α-helix结构。半定量RT-PCR结果表明CiLGP2基因的mRNA在各组织中均有表达,脾脏、皮肤、心脏和肠的表达量较高,注射GCRV后表达量上调。脾脏中12 h的表达量显著上升(14.50倍,P<0.05),24 h达到峰值(19.00倍,P<0.05),48 h后稍有下降(10.40倍)并在后期维持该水平(P<0.05)。肝脏中24 h表达量显著上升(3.80倍,P<0.05),48 h达到峰值(10.70倍,P<0.05),72 h下降(5.80倍,P<0.05)并在后期维持高表达(P<0.05)。以上试验结果提示CiMDA5和CiLGP2基因的表达和草鱼抗病毒先天性免疫存在一定的联系,为进一步研究鱼类RLR信号途径奠定了基础。深入了解这两个重要的胞浆病毒识别受体将有助于寻找新的有效手段来预防和控制水产病毒性疾病的感染。更多还原

【Abstract】 Grass carp Ctenopharyngodon idella is considered an economically important aquaculture species in China with the largest production among the“Great Four Cultured Fish”. Despite its fast growth, delicate taste and low production cost, the toughest challenge is that grass carp is susceptible to a wide variety of diseases, and the annual loss reaches billion yuan. Grass carp hemorrhage caused by Grass Carp Reovirus (GCRV) is accused of a threat to grass carp health. However, the available vaccines are subject to be less effective because of the primitive specific immunity of grass carp. Thus, identifying critical molecules in the innate immunity networks will become the basis for disease-resistant breeding in the future.In the phylogenetic history, a number of homologs executing antiviral roles are revolutionarily conserved in metazoans. The remarkable examples include: TLR (Toll-like receptors) and RLR (RIG-I-like receptors) signaling pathways. The RLR pathways have recently been recognized as a critical antiviral mechanism in mamamals. There are three members referred to as RLRs: RIG-I (Retinoic acid-inducible gene-I), MDA5 (Melanoma differentiation-associated gene 5) and LGP2 (Laboratory of genetics and physiology 2), which play separate roles in recognition of cytosolic nuclear acids and activate a series of host immune response to viruses. It is thereby significant to uncover the roles of fish RLRs in combating aquatic viruses.In the present study, degenerate primers were designed based on sequence alignement to produce core amplicons, followed by RACE (Rapid amplification of cDNA end), semi-quantitative RT-PCR (sqRT-PCR), quantitative reat-time PCR as well as bioinformatic analysis in succession. The complete cDNA of CiMDA5 and CiLGP2 genes was cloned to explore their roles in anti-GCRV and the phylogeny of RLRs. This research provides new insights to the unknown fish RLR family. Results are as follows:(1) Molecular cloning and immune responsive expression of CiMDA5 geneThe cytoplasmic helicase protein MDA5 recognizes long molecules of viral double-stranded RNA (dsRNA) and single-stranded RNA with 5’ triphosphate and mediates type I interferon secretion. In the present study, the first MDA5 gene in fish was cloned and characterized from grass carp. The full length of CiMDA5 cDNA comprises 3233 nucleotides in length and encodes a polypeptide of 961 amino acids. The deduced amino acid sequence contained six main structural domains: a CARD (Caspase activation and recruitment domain), a DEXDc (DEAD/DEAH box helicase domain), a ResIII (Conserved restriction domain of bacterial type III restriction enzyme), two HELICc (Helicase superfamily c-terminal domain) and a RD (Regulatory domain). The CiMDA5 mRNA was widespread expression in the tested tissues, was high level in spleen, skin and gill tissues, and was up-regulated by GCRV injection by sqRT-PCR assay. The CiMDA5 transcripts in spleen were significantly up-regulated at 12 h (1.80 folds, P<0.05), reached the crest at 24 h (7.48 folds, P<0.05), and then recovered to normal level at 48 h post-injection (P>0.05) of GCRV. In liver, the temporal expression of CiMDA5 mRNA was significantly increased at 48 h (5.00 folds, P<0.05) and returned to control level at 72 h (P>0.05) after GCRV challenge.(2) Identification and expression profiling analysis of CiLGP2 cDNALGP2 is homogenous to RIG-I and MDA5 without a CARD required for signal transduction, plays a pivotal role in modulating signaling by RIG-I and MDA5 for interferon synthesis. In this study, a novel LGP2 gene from grass carp designated as CiLGP2 was isolated and characterized. The full-length cDNA of CiLGP2 was of 2920 bp with five instability motifs (ATTTA). The open reading frame was of 2043 bp and encoded a polypeptide of 680 amino acids, including five main overlapping structural domains: two DEXDc, one ResIII, one HELICc and one RD. There was one moreα-helix in the RD, compared with that in human. The CiLGP2 mRNA was ubiquitous expression in the tested tissues, was high level in spleen, skin, heart and intestine tissues, and was up-regulated by GCRV injection by sqRT-PCR assay. The CiLGP2 expression in spleen was significantly up-regulated at 12 h (14.50 folds, P<0.05), reached the crest at 24 h (19.00 folds, P<0.05), and then dropped a little at 48 h (10.40 folds) post-injection of GCRV and kept this level till the following test period (P<0.05). In liver, the temporal expression of CiLGP2 mRNA was significantly increased at 24 h (3.80 times, P<0.05), reached peak at 48 h (10.70 times, P<0.05), and then decreased a little bit at 72 h (5.80 times, P<0.05) and kept this high level till the test finish (P<0.05).These data implied that the expression of CiMDA5 and CiLGP2 genes is associated with the antiviral innate immune defense to GCRV; meanwhile, the results lay a foundation for the further research of RLR signaling pathways in fish. These two cytoplasmic receptors are critical in virus recognition; in-depth understanding of them will be conducive to the development of effective measures against viral infection in aquaculture.更多还原