【作者】 兰江风；

【导师】 王金星；

【作者基本信息】 山东大学 ， 动物学， 2014， 博士

【摘要】 甲壳动物是重要的经济养殖动物,甲壳动物的养殖与捕捞量占世界水产品总量的百分之十。然而,近年来水产病害频发使水产养殖业蒙受了巨大的损失。为了防控甲壳类水产养殖中病害的发生,对它们先天免疫机理的深入研究是很有必要的。虽然经过近20年的不懈努力,甲壳动物先天免疫的研究取得了显著的成果,但距离真正解决水产病害这一难题还有一定的距离,很多先天免疫的分子机制还有待揭示。本论文中,作者从克氏原螯虾中鉴定出一种抑制素,发现其能够抑制病毒复制。还初步探讨了对虾JAK/STAT信号通路的组成以及在对虾细菌免疫中的功能与调控。1.克氏原螯虾抑制素参与抗病毒免疫的研究抑制素(Prohibitins,PHBs)是一类在真核生物中广泛表达的,具有很高保守性的蛋白质。它与细胞凋亡、癌症、细胞死亡、应激反应、细胞增殖以及免疫调控等众多生命反应有关。但是,PHB在甲壳动物免疫中的功能报道还很少。本研究中从克氏原螯虾中鉴定出一种抑制素,生物信息学分析确定其为抑制素1,命名为PcPHB1。PcPHB1在小龙虾各组织中广泛表达,在白班综合征病毒(WSSV)刺激后其在核酸水平和蛋白水平的表达量都有明显的提高。研究发现,在病毒刺激下,与对照组相比PHB1重组蛋白能够明显的降低克氏原螯虾的死亡率。同时,当用RNA干扰的方法在龙虾体内敲降PHB1的表达,并感染WSSV后,克氏原螯虾的死亡率明显增加。拯救实验(即在PHB1干扰后,向克氏原螯虾体内注入PHB1重组蛋白,之后再使克氏原螯虾感染病毒)表明,PHB1重组蛋白能够减少克氏原螯虾的死亡率。为了进一步研究PHB1抗病毒免疫的分子机制,我们检测了PHB1与病毒蛋白的相互作用。Pull-down和Far-western等实验表明,PHB1可能同病毒的囊膜蛋白VP24.VP26和VP28相互作用。据报道VP28参与病毒侵染宿主细胞的过程,能够帮助病毒进入宿主细胞。VP26在病毒装配过程中发挥了重要作用,而VP24在病毒感染的早期发挥作用。因此,PHB1可能通过与病毒囊膜蛋白VP28、VP26和VP24的相互作用,抑制病毒的侵染和病毒感染后病毒的组装。这是甲壳动物PHB1参与抗病毒免疫的首次报道。2.日本对虾信号转导与转录激活信号通路(JAK/STAT)参与细菌免疫的研究JAK-STAT信号通路是一条由细胞因子刺激的信号转导通路,参与细胞的增殖、分化、凋亡以及免疫调节等许多重要的生物学过程。该信号通路的传递过程相对简单,它主要由三个成分组成,即酪氨酸激酶相关受体、酪氨酸激酶JAK和转录因子STAT。近年来发现细胞因子信号转导抑制子(Suppressor of cytokine signaling, SOCS)是一类STAT诱导表达的STAT的抑制因子(SSI),它能够负调控JAK/STAT信号通路。在果蝇中,JAK/STAT信号通路与其发育和细胞及体液免疫反应有关。在对虾中,有报道称在ploy IC以及PGN刺激下,STAT能够被诱导上调表达。在凡纳滨对虾中,WSSV刺激下STAT发生磷酸化,并且STAT磷酸化后进入细胞,调控效应分子的表达。JAK/STAT信号通路在甲壳动物中参与细菌免疫反应的报道很少,其参与抗细菌免疫的分子机制还有待阐明。在本研究中,我们鉴定了日本囊对虾JAK/STAT通路的四种成分,包括膜受体MjDome,膜受体配体MjIL-CTL,信号转导与转录激活子MjSTAT和细胞因子信号通路抑制因子MjSOCS2。它们有类似的组织分布,都在心、腮和肠中有较高的表达水平。MjSTAT和MjSOCS2在弧菌刺激后有上调表达的趋势。为了研究该途径的功能,作者首先建立了对虾中该途径的标志：弧菌刺激能够引起STAT的磷酸化。进一步研究发现弧菌表面分子脂多糖(LPS)可以诱发STAT的磷酸化过程。同时,本研究鉴定到LPS的可能的受体(MjIL-CLT)介导了LPS与JAK/STAT信号通路的受体Dome的结合。我们用LPS作为诱导剂,筛选出五种被JAK/STAT通路特异性调控的抗菌肽,包括ALF4、ALF5、ALF6、Cru3和Cru4。STAT被干扰后,对虾对细菌的清除能力减弱,对虾的死亡率增加。作者还对JAK/STAT途径的调控进行研究,发现SOCS2能够抑制STAT的磷酸化过程。SOCS2被干扰后,对虾对细菌的清除能力增强,对虾的死亡率减少。这些结果表明,JAK/STAT信号通路通过调节抗脂多糖因子和甲壳肽等抗菌肽的表达参与细菌免疫过程。3.日本对虾一种含有跨膜结构域的甲壳肽参与细菌免疫的研究甲壳肽(Crustins)是甲壳动物中特有的、含有乳清酸蛋白结构域、一般具有抑制蛋白酶和抑制细菌活性的抗菌肽。在本研究中,我们从日本囊对虾中克隆并鉴定了一种新型的甲壳肽,它含有一个信号肽,一个跨膜结构域和一个乳清酸蛋白(whey acidic protein, WAP)结构域,命名为MjMCru。MjMCru在心脏、鳃、胃和肠中有较高的表达水平,在血细胞和肝胰腺中表达量较低。在弧菌和金黄色葡萄球菌刺激后MjMCru在血细胞中的表达量明显增加。在对虾体内注射MjMCru的WAP多肽,能够有效的减少弧菌和金黄色葡萄球菌刺激下对虾的死亡率。进一步研究发现,MjUCru能够通过同细菌表面多糖分子(如LPS、PGN和LTA)结合,而结合于细菌表面。在对虾体内注射WAP多肽能够增强对虾血细胞对弧菌和金黄色葡萄球菌的吞噬作用。这些结果表明,MjMCru在抗细菌免疫中发挥着重要作用。更多还原

【Abstract】 Crustaceans are important economic creatures, and crustaceans farming and fishing accounts for twenty percent of the world economy aquatic organisms. However, in recent years, aquaculture of aquatic diseases caused massive economic losses. The study of the crustacean innate immunity necessary, it was benefit for prevention and control of aquatic diseases, as well as the mechanism of immunity.In the present study, we identified a prohibitin from red swamp crayfish and found that it can inhibit WSSV replication. We have studied the mechanisms of signal transduction and transcriptional activity signaling pathways, and we explore the role of STAT signaling pathway in bacteria immunity. We have cloned a crustin which with a transmembrane domain, and found it involved in phagocytosis.1. Prohibitin1prevents infection of WSSV in red swamp crayfishProhibitins (PHBs) are ubiquitously expressed conserved proteins in eukaryotes that are associated with apoptosis, cancer formation, aging, stress responses, cell proliferation, and immune regulation. However, the function of PHBs in crustacean immunity remains unknown. In the present study, we identified a PHB in red swamp crayfish Procambarus clarkii, which was designated as PcPHB1. PcPHB1was widely distributed in several tissues and its expression was significantly upregulated by white spot syndrome virus (WSSV) challenge at the mRNA level and the protein level. These observations prompted us to investigate the role of PcPHB1in the crayfish antiviral response. Recombinant PcPHB1significantly reduced the amount of WSSV in crayfish and the mortality of WSSV-infected crayfish. The quantity of WSSV in PcPHB1knockdown crayfish was increased compared with the controls. The effects of RNA silencing were rescued by rPcPHB1re-injection. We further confirmed the interaction of PcPHB1with the WSSV envelope proteins VP28, VP26, and VP24using pull-down and far-Western overlay assays. Finally, we observed that the colloidal gold-labeled PcPHB1was located on the outer surface of the WSSV, which suggests that PcPHB1specifically binds to the envelope proteins of WSSV. VP28, VP26, and VP24are structural envelope proteins and are essential for attachment and entry into crayfish cells. Therefore, PcPHB1exerts its anti-WSSV effect by binding to VP28, VP26, and VP24, preventing viral infection. This study is the first report on the antiviral function of PHB in the innate immune system of crustaceans.2. JAK/STAT signaling pathway associated with antibacterial response in shrimpJAK/STAT pathway was reported involved in immune response of anti-bacterial and anti-viral immune responses. The suppressor of cytokine signaling proteins (SOCS) is a class of STAT induced STAT inhibitor (SSI), which negatively regulates the JAK/STAT signaling pathway. In shrimp, STAT was up-regulated expression in challenge with ploy IC and PGN. WSSV could induce the increase expression of STAT, and it induced the phosphorylation of STAT in shrimp. When the STAT was phosphorylation, it would translocation to the nuclear of cell and regulated the expression of effectors. However, the molecular mechanisms of STAT involved in bacterial immune are not clear in shrimp. In the present study, we found that the cell wall molecule of Vibrio anguillarum could induce the phosphorylation of STAT. SOCSs are feedback inhibitors of STAT pathway, SOCS1and SOCS3reported inhibiting the phosphorylation of STAT, and SOCS2had been reported to participate in NF-κB signaling pathways. Although there have been reports that SOCS2can be up-regulatedby ploy IC and PGN, which indicating that SOCS2may be involved in STAT phosphorylation process. However, there no report showed that SOCS2could inhibit the phosphorylation of STAT. In the present study, we found LPS could induce the phosphorylation of STAT. The special lectin MjIL-CT-L, which with IL similar domain, could bind to LPS and the ILR domain of Dome. And when Dome was knocked down, the phosphorylation of STAT was inhibited in challenge with LPS. SOCS2is able to feedback inhibition the process of STAT phosphorylation.We screened out some AMPs, which were induced expression by LPS specially. When STAT was knocked down in vivo, the bacterial clearance level was lower than the control, and the mortality of shrimp infected with bacteria was increase comparing with control. When SOCS2was knocked down in vivo, the bacterial clearance level was higher than the control, and the mortality of shrimp challenge with bacteria was decrease. These results indicate that STAT pathway involved in antibacterial immune response by regulating the expression of antimicrobial peptides in shrimp.3. A novel crustin from Marsupenaeus japonicus promotes hemocyte phagocytosisThe crustins, one kind of antimicrobial peptides (AMPs) from crustacean with a whey acidic protein (WAP) domain have functions in protease inhibition and antimicrobial activity. In the percent study, a novel crustin with a single WAP domain and a transmembrane region was found in kurume shrimp, Marsupenaeus japonicus, and designated as MjMCru. Quantitative real-time PCR (qRT-PCR) analysis showed that MjMCru was mainly expressed in heart, gills, stomach and, and low level expression in hemocytes and hepatopancreas. The expression level of MjMCru was up-regulated in hemocytes challenge with bacteria. Injection of WAP peptide could increase the survival rate of shrimp infected with V. anguillarum and S. aureus. Further study found that MjMCru could bind to bacteria by binding to the bacteria cell wall molecules such as LPS, PGN and LTA. MjMCru (WAP) could increase the phagocytic rate of hemocytes in shrimp infected with V. anguillarum and S. aureus. These results suggested that MjMCru had an important role in antibacterial immunity of shrimp.更多还原