【作者】 于爱清；

【导师】 王群；

【作者基本信息】 华东师范大学 ， 水生生物学， 2014， 博士

【摘要】 中华绒螯蟹(Eriocheir sinensis)是我国重要的经济养殖蟹类之一,又俗称河蟹或大闸蟹,隶属于节肢动物门(Athropoda)、软甲纲(Malacostraca)、十足目(Decapoda)、方蟹科(Grapsidae)、绒螯蟹属(Eriocheir)、中华绒螯蟹种(Eriocheir sinensis),不仅肉味鲜美,营养丰富,而且是我国久负盛名的美食,因此具有极高的经济和养殖价值。红螯光壳螯虾(Cherax quadricarinatus),又俗称澳洲淡水龙虾或者红螯螯虾,隶属于甲壳纲(Crustacea)、十足目(Decapoda)、拟螯虾科(Parastacidae)、光壳虾属(Cherax),原产于澳大利亚,于1991年首次引入我国试养,并于次年开展人工繁殖和自然繁殖获得成功,因其具有肉质细嫩、生长速度快、养殖产量高、富含低胆固醇等特点而被广泛推广养殖。伴随着两种甲壳动物养殖规模的扩大以及集约化养殖的发展,各种养殖病害频发,严重制约了这两种甲壳动物养殖产业的发展。而这两种甲壳动物,均凭借其独特的先天性免疫系统,在外界不良生存环境中有效地抵御外界病原菌的入侵,因而其先天性免疫防御机制的研究备受关注。有鉴于此,本研究以这两种甲壳动物为研究对象,开展了对这两种甲壳动物免疫相关基因的功能研究,以期能够完善甲壳动物先天性免疫防御机制的理论研究,为制定有效地病害防御策略提供坚实的理论基础与支持。本研究主要研究内容与结果如下：1)中华绒螯蟹Toll基因的克隆与表达分析Toll样受体在诱导病原菌的先天性免疫应答反应中起着重要作用,因此在大部分昆虫中均已经有所报道,但是在中华绒螯蟹中却未见报道。在本文中,我们克隆了两个新的中华绒螯蟹Toll基因：EsTolll和EsToll2。EsTolll基因的cDNA全长3963bp,开放阅读框3042bp,编码1013个氨基酸。其胞外区主要由17个亮氨酸重复序列组成,胞内区主要由139个氨基酸残基的TIR结构域组成。EsToll2基因cDNA全长4419bp,开放阅读框2667bp,编码888个氨基酸。其胞外区主要由10个亮氨酸重复序列组成,胞内区主要由139个氨基酸残基的TIR结构域组成。聚类分析的结果显示,EsToll1和EsToll2首先与其它甲壳动物的Toll受体家族聚为一支,然后与其它节肢动物的Toll受体家族聚为一支。荧光定量PCR(qRT-PCR)分析结果表明：EsTolll和EsToll2在各组织中都能够检测到,经由脂多糖(LPS)、肽聚糖(PG)和葡聚糖(GLU)免疫刺激后,表达量具有不同程度的变化。比较重要的是,EsToll2mRNA的表达量几乎在所有免疫时间段内均有显著的增加。综上,EsTolll和EsToll2对于不同的病原相关分子的刺激具有不同的可诱导性,对中华绒螯蟹先天性免疫应答的研究具有重要意义。2)中华绒螯蟹Tube基因的克隆与表达分析Tube作为Toll信号通路中的重要组分,在机体存活、发育以及先天性免疫等生理活动中具有重要的作用。目前已经在对虾中被成功识别,但是未见在中华绒螯蟹中有所报道。在本文中,我们成功克隆得到了中华绒螯蟹Tube基因,简称为EsTube。EsTube基因cDNA全长2247bp,开放阅读框1539bp,编码512个氨基酸。EsTube蛋白主要包括一个由116个氨基酸残基组成的死亡结构域(death domain, DD)和一个由272个氨基酸残基组成的丝组氨酸蛋白激酶结构域(S TKc)。聚类分析的结果显示,EsTube首先与其它无脊椎动物Tube和Tube样蛋白聚为一支,然后与脊椎动物的IRAK-4蛋白聚为一支,最后与其它无脊椎动物Pelle蛋白聚为一支。实时荧光定量PCR (qRT-PCR)的结果显示,EsTube高度表达在卵巢和精巢,中度表达在胸神经节和胃；在所有发育阶段均能检测到EsTube的表达,高度表达在精巢发育的精细胞阶段(十月份)和卵巢发育的Ⅲ-2阶段(十一月份)；在不同病原相关分子模式(LPS、β-1,3-glucan and PG)的免疫刺激下,具有不同的诱导表达水平。综上,本研究的结果表明,EsTube在中华绒螯蟹免疫和发育方面具有重要的作用,具有免疫和发育的双重功能。3)中华绒螯蟹Dorsal基因的克隆与表达分析Dorsal作为Toll信号通路中的重要组分,在先天性免疫应答的诱导和识别中具有重要作用。在本文中,我们在中华绒螯蟹中克隆了一个NF-κB样核转录因子—Dorsal,简称为EsDorsal。EsDorsal基因cDNA全长2493bp,开放阅读框2022bp,编码673个氨基酸。EsDosal蛋白主要包括一个由171个氨基酸组成的RHD结构域和一个由102个氨基酸组成的IPT结构域。聚类分析的结果显示,EsDorsal首先与其它无脊椎动物的Dorsals或者NF-κBs聚为一支,然后与脊椎动物的NF-κBs聚为一支。实时荧光定量PCR(qRT-PCR)的结果显示,EsDorsal在免疫相关组织中表达量较高,经过脂多糖(LPS)、肽聚糖(PG)和葡聚糖(GLU)免疫刺激后,表达量具有不同程度的变化。重要的是,相对于对于肽聚糖(PG)和葡聚糖(GLU), EsDorsal对脂多糖(LPS)的免疫刺激更加敏感。综上,EsDorsal对于不同的病原相关分子的刺激具有不同的可诱导性,对中华绒螯蟹先天性免疫应答机制的研究具有重要意义。4)红螯光壳螯虾Crustin基因的克隆与功能分析抗菌肽作为一种重要的免疫效应因子,对于红螯光壳螯虾(Cherax quadricarinatus)先天性免疫应答防御反应具有重要作用。本研究综合同源克隆和SMART-RACE技术,从红螯光壳螯虾的血细胞中成功克隆得到了一个重要的抗菌肽Crustin,简称为CqCrustin。CqCrustin基因cDNA全长为608bp,开放式阅读框为324bp,编码107个氨基酸,在其推测的氨基酸序列的N端含有一个由16个氨基酸残基组成的信号肽,C端含有一个由48个氨基酸残基组成的乳清酸蛋白(WAP)结构域。基于其WAP结构域的序列信息,进行聚类分析的结果显示,CqCrustin首先与其它甲壳动物的Ⅰ型Crustin聚为一支,然后与其它甲壳动物Ⅱ型Crustin聚为一支,最后于Ⅲ型Crustin聚为一支。多序列比对的结果显示,CqCrustin与克氏原螯虾(Procambarus clarkii) PcCrustin2具有较高的序列相似性(69%)。实时荧光定量PCR (qRT-PCR)的结果显示,CqCrustin主要表达在免疫相关组织,且在红螯光壳螯虾血细胞中的表达量最高；利用LPS,GLU和PG三种病原相关分子模式免疫刺激后,CqCrustin的mRNA表达水平在红螯光壳螯虾血细胞中具有不同程度的显著上调。为了进一步研究其生物学活性,利用原核表达技术获得的重组蛋白rCqCrustin对于不同的病原菌具有不同程度的结合活性,而且对不同的病原菌的生长速度亦具有不同程度的抑制作用。综上可见,CqCrustin在红螯光壳螯虾先天性免疫识别和防御过程中具有重要作用。5)红螯光壳螯虾Dscam基因的克隆与功能分析细胞粘附分子(Dscam)属于免疫球蛋白超级家族中的重要一员,在无脊椎动物,特别是节肢动物的先天性免疫识别和防御中具有重要作用。本研究综合同源克隆和SMART-RACE技术,从红螯光壳螯虾(Cherax quadricarinatus)的血细胞中成功克隆得到了其Dscam基因,简称为CqDscam。GqDscam蛋白含有类似于其它无脊椎动物的Dscam蛋白同系物的典型结构域,主要包括一个信号肽、10个免疫球蛋白结构域(Ig)、6个Ⅲ型纤连蛋白结构域(FNⅢ)、一个跨膜区和一个细胞质尾结构域。聚类分析的结果显示,CqDscam蛋白首先与其它甲壳动物的Dscam同系物聚为一支,然后与其它无脊椎动物的Dscam同系物聚为一支,最后与脊椎动物的Dscam同系物聚为一支。胞外结构域的检测结果表明,CqDsccam蛋白的N端具有多个可变剪切亚型,其中Ig2结构域具有30个亚型,Ig3结构域具有33个亚型,Ig7具有19个亚型,跨膜区具有2个亚型,理论上,总共可产生多达37620个不同的亚型。qRT-PCR的检测结果表明,CqDscam组成性表达在红螯螯虾各组织,特别是神经和免疫相关的组织：利用脂多糖(LPS)、肽聚糖(PG)和β-1,3-葡聚糖(GLU)免疫刺激红螯螯虾后,CqDscam的mRNA表达水平具有不同程度的诱导上调。CqDscam重组蛋白的功能检测显示,rCqDscam能够不同程度地识别不同的细菌,并且能够不同程度地抑制革兰氏阳性菌(金黄色葡萄球菌)和革兰氏阴性菌(大肠杆菌)的生长。此外,免疫荧光的结果显示,CqDscam主要分布于红螯螯虾血细胞的表面。综上可见,CqDscam在红螯螯虾的先天性免疫识别和防御中具有重要作用。更多还原

【Abstract】 The Chinese mitten crab(Eriocheir sinensis) as one of the commercially important crab, also was known as the fresh water crab、hairy crab and so on, belongs to Athropoda, Malacostraca, Decapoda, Grapsidae, Eriocheir, Eriocheir sinensis. The Chinese mitten crab not only is prestigious food, but also has better nutritive value and raising value because of its rich-nutrition and delicious taste. The Red claw crayfish (Cherax quadricarinatus), that also is known as the Australian freshwater lobster or red claw crayfish, belongs to Crustacea, Decapoda, Parastacidae. Cherax quadricarinatus, that is native to Australia and introduced into China in1991for the first time. It is breeding successfully and widely cultured in the next year because of its fast growth, delicious taste, high yield and adaptability. With the development of the intensive aquaculture and the enlargement of the cultivation scale, the frequent outbreaks of diseases have caused decreased production and catastrophic losses in the past decade. Crustacean can induce rapid and effective immune responses to clear the intruding pathogens relying largely on innate immunity, which attracts more and more attention. Upon this backdrop, studying the structure and transcriptional responses of potential immune-related genes may facilitate a better understanding of the crustacean immune defense and recognition mechanisms and support the sustainable development of better disease management strategies in the crustacean farming industry.The main research contents and results are as follows:1) Two novel Toll genes(EsTolll and EsToll1) from Eriocheir sinensis are differentially induced by lipopolysaccharide, peptidoglycan and zymosanTolls/Toll-like receptors (TLRs) play an essential role in initiating innate immune responses against pathogens and are found throughout the insect kingdom but have not yet been reported in the crustacean, Eriocheir sinensis. For this purpose, we cloned two novel Toll genes from E. sinensis, EsTolll and EsToll2. The full-length cDNA of EsTolll was3,963bp with a3,042-bp open reading frame (ORF) encoding a1,013-amino acid protein. The extracellular domain of this protein contains17leucine-rich repeats (LRRs) and a139-residue cytoplasmic Toll/interleukin-1receptor (TIR) domain. The cDNA full-length of EsToll2was4,419bp with a2,667-bp ORF encoding an888-amino acid protein with an extracellular domain containing10LRRs and a139-residue cytoplasmic TIR domain. By phylogenetic analysis, EsTolll and EsTolll clustered into one group together with Tolls from other crustaceans. Quantitative RT-PCR analysis demonstrated that a) both EsTolll and EsToll2were constitutively expressed in all tested crab tissues; b) EsTolll and EsToll2were differentially induced after injection of lipopolysaccharides (LPS), peptidoglycan (PG) or zymosan (GLU). Importantly, EsToll2expression was significantly upregulated at almost all time intervals post-challenge with LPS, PG and GLU. Our study indicated that EsTolll and EsToll2are differentially inducibility in response to various PAMPs, suggesting their involvement in a specific innate immune recognition mechanism in E. sinensis.2) Molecular cloning and expression analysis of Tube from Eriocheir sinensisAs a key component of the Toll signaling pathway, Tube plays central roles in many biological activities, such as survival, development and innate immunity. Tube has been found in shrimps, but have not yet been reported in the crustacean, Eriocheir sinensis. In this study, we cloned the full-length cDNA of the adaptor Tube for the first time from E sinensis and designated the gene as EsTube. The full-length cDNA of EsTube was2,247-bp with a1,539-bp open reading frame (ORF) encoding a512-amino acid protein. The protein contained a116-residue death domain (DD) at its N-terminus and a272-residue serine/threonine-protein kinase domain (S_TKc) at its C-terminus. Phylogenetic analysis clustered EsTube initially in one group with other invertebrate Tube and Tube-like proteins, and then with the vertebrate IRAK-4proteins, finally with other invertebrate Pelle proteins. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis results showed that EsTube was highly expressed in ovary and testis, and moderately expressed in thoracic ganglia and stomach. EsTube was expressed at all selected stages and was highly expressed in the spermatid stage (October, testis) and the stage III-2(November, ovary). EsTube was differentially induced after injection of lipopolysaccharides (LPS), peptidoglycan (PG) or zymosan (β-1,3-glucan). Our study indicated that EsTube might possess multiple functions in immunity and development in E. sinensis.3) Molecular cloning and expression analysis of a dorsal homologue from Eriocheir sinensisDorsal as a crucial component of Toll signaling pathway, played important roles in induction and regulation of innate immune responses. In this study, we cloned a NF-κB-like transcription factor Dorsal from Eriocheir sinensis and designated it as EsDorsal. The full-length cDNA of EsDorsal was2,493bp with a2,022-bp open reading frame (ORF) encoding a673-amino acid protein. This protein contained a171-residue conserved Rel homology domain (RHD) and a102-residue Ig-like, plexins and transcription factors domain (IPT). By phylogenetic analysis, EsDorsal was clustered into one group together with other invertebrate Dorsals or NF-κBs, and then clustered with vertebrate NF-κBs. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis results showed that a) EsDorsal had higher expression level in immune organs; b) EsDorsal differentially induced after injection of lipopolysaccharides (LPS), peptidoglycan (PG) or zymosan (GLU). Importantly, EsDorsal was more responsive to LPS than GLU and PG. Collectively, EsDorsal was differentially inducibility in response to various PAMPs, suggesting its involvement in a specific innate immune regulation in E. sinensis.4) A novel Crustin involves in antibacterial responses in the red claw crayfish, Cherax quadricarinatus Antimicrobial peptides are important immune effectors and play important roles in mediating innate immune responses against intruding pathogens. Here, we successfully isolated and characterized a novel Crustin from Red claw crayfish Cherax quadricarinatus and named it as CqCrustin. The deduced amino acid sequence of CqCrustin exhibited the typical configuration similar to other crustacean Crustin orthologs, including one signal peptide region at N-terminus between1and16and a long whey acidic protein domain (WAP domain) at C-terminus between60and107along with a WAP-type "four-disulfide core" motif. Phylogenetic analysis showed that CqCrustin was clustered with other crustacean Type Ⅰ Crustins firstly, and then with other crustacean Type Ⅱ Crustins, finally with other crustacean Type Ⅲ Crustins. CqCrustin showed higher sequence similarity (69%) with Crustin2from Procambarus clarkii (Pc-Crustin2). Transcription of CqCrustin-Ⅰ were both1) detected in all tissues, especially in hemocytes and gill; b) differentially induced in hemocytes post β-1,3-glucans (GLU), lipopolysaccharides (LPS) and peptidoglycans (PG) injection at selected time points. To understand its biological activity, the recombinant CqCrustin protein was constructed and expressed in Escherichia coli BL21(DE3). Recombinant protein rCqCrustin exhibited distinct bacterial binding activities against different Gram-positive bacteria, Gram-negative bacteria and fungus. Furthermore, bacterial growth inhibition assays demonstrated that rCqCrustin responds positively to the growth inhibition of different Gram-positive bacteria, Gram-negative bacteria and fungus. These findings suggested CqCrustin may involve in a specific innate immune recognition and defense mechanism against bacteria and fungus in Cherax quadricarinatus.5) Identification and characterization of Dscam isoforms isolated from the red claw crayfish Cherax quadricarinatusThe Down syndrome cell adhesion molecule (Dscam) belongs to the immunoglobulin superfamily (IgSF) member and has been identified and isolated from some vertebrates and invertebrates. Recently, many studies have confirmed the important role of Dscam in mediating innate immune response against intruding pathogens and wiring of the nerve system in invertebrates, especially in Arthropod. Here, we successfully isolated and characterized the Dscam from Red claw crayfish Cherax quadricarinatus and named it as CqDscam. The deduced amino acid sequence of CqDscam exhibited the typical configuration similar to other invertebrate Dscam orthologs, including one signal peptide,10immunoglobulin (Ig) domains,6fibronectin type III (FNIII) domains and one transmembrane (TM) domain and cytoplasmic tail domain. Phylogenetic analysis showed that CqDscam was clustered with other invertebrate Dscams firstly, and then with vertebrate Dscams. In the extracellular region, the variable regions of CqDscam were located in N-terminal half of Ig2and Ig3domains and the complete Ig7domain. The CqDscam extracellular variants and transmembrane domain variants were produced by mutually exclusive alternative splicing events and could generate more than37,620different unique isoforms. Transcription of CqDscam were both1) detected in all tissues, especially in immune tissues and nerve tissues; b) differentially induced in hemocytes post glucans (GLU), lipopolysaccharides (LPS) and peptidoglycans (PG) injection at selected time points. Importantly, we had detected membrane-bound and secreted Dscam isoforms in C. quadricarinatus and showed that secreted isoforms were extensively transcribed post different PAMPs challenge, respectively. Results from immuno-localization assay revealed that CqDscam evenly distributed in the cell surface of hemocytes. In addition, the PAMPs specific isoforms of CqDscam were shown to be associated with bacterial clearance and phagocytosis in Red claw crayfish. These findings suggested CqDscam may involve in a specific innate immune recognition and defense mechanism in Cherax quadricarinatus.更多还原