【作者】 赵大显；

【导师】 陈立侨；

【作者基本信息】 华东师范大学 ， 动物学， 2009， 博士

【摘要】 中华绒螯蟹(Eriocheir sinensis)是我国的重要增养殖对象之一。近年来,随着其养殖规模的不断扩大和集约化程度的提高,由细菌、病毒和类立克次体生物等引起的各种病害日趋严重,给中华绒螯蟹养殖业造成了巨大的损失。至今,病害仍然是困扰其养殖业的一个重要因素。除改善环境因素外,提高机体自身免疫力和抗病力是解决这一难题的一个重要方法。本论文试图从分子角度来研究中华绒螯蟹的免疫防御机制,以其为这一重要方法提供坚实的理论基础。本研究借助分子生物学和生物信息学手段,首次完成对中华绒螯蟹血细胞cDNA文库的构建和表达序列标签(EST)的分析,不仅丰富了中华绒螯蟹的基因数据,同时筛选出许多与免疫相关的基因序列。在此基础上,通过cDNA末端快速扩增(RACE)和实时定量PCR技术进一步对主要的免疫相关基因进行了克隆和表达模式分析,这些研究为了解中华绒螯蟹免疫机制提供了基础资料,同时也为蟹病的防治提供了新思路。主要研究结果如下:1采用常规cDNA文库构建技术,成功构建了滴度为4.50×10~6CFU/mL,库容为3.3×10~6CFU/mL,重组率为73%的中华绒螯蟹血细胞cDNA文库;从文库中随机挑取了3,118个克隆进行5’-端单向测序,经去除低质量和污染序列后,共得到3,041条高质量EST序列,这些序列的平均长度为561bp。经拼接后获得了1,049条unique序列,包括292条重叠群(Contig)和757条单一序列(Singleton)。通过比对发现有488条Unique序列被注释成已知的功能基因,551条Unique序列未被注释任何功能;通过Gene Ontology功能分类发现了26个中华绒螯蟹先天性免疫反应,如参与酚氧化酶系统的丝氨酸蛋白酶、Kazal-型蛋白酶抑制剂、丝氨酸蛋白酶抑制剂、α-巨球蛋白、酚氧化酶原激活因子以及酚氧化酶酶原,参与凝集反应的谷胱酰胺转移酶,与抗氧化系统相关的超氧化物歧化酶、谷胱甘肽转移酶以及谷胱甘肽过氧化物酶,抗菌肽类的抗脂多糖因子、甲壳素、以及Osmotin,模式识别分子类的C-型凝集素、甘露糖结合蛋白、脂多糖和β-1,3葡聚糖结合蛋白、Toll受体蛋白。通过EST表达丰度分析发现,中华绒螯蟹血细胞主要免疫相关基因为Kazal-型蛋白酶抑制剂和C-型凝集素。2模式识别蛋白及其功能的研究是当前甲壳动物免疫学的研究重点之一,许多物种的模式识别蛋白及其功能已经明确。本研究根据EST提供的信息,采用RACE技术成功克隆了中华绒螯蟹一种模式识别蛋白-脂多糖和β-1,3葡聚糖结合蛋白(LGBP)基因全长cDNA序列。生物信息学分析发现,该序列全长为1,236bp,包括26 bp的5’-末端非转录区(5’-UTR),1,089 bp的开放阅读框(ORF)和121bp的3’-末端非转录区(3’-UTR),共编码362个氨基酸,包含一个由15个氨基酸组成的信号肽;蛋白序列与中国明对虾(Penaeus chinensis)、细角滨对虾(Litopenaeus stylirostris),凡纳滨对虾(Litopenaeus vannamei)和淡水螯虾(Pacifaxtacus leniusculus)LGBP的相似性分别为67%、66%、66%、61%;该基因编码蛋白含有几个保守的区域,如整合素结合区,激酶C磷酸化位点,N—连接的糖基化位点等,另外还发现了一个水解酶位点。实时定量PCR结果显示,LGBP基因mRNA在中华绒螯蟹肝胰腺、鳃、肌肉、血细胞、胃和肠中均有表达,其中在血细胞中的表达量最高。用嗜水气单胞菌进行急性感染实验发现,LGBP在血细胞中的表达量在感染后1.5 h达到最高水平,与对照组相比存在显著性差异(P＜0.05),而后表现为下降趋势,在感染后12 h恢复到起始水平,说明该基因参与了中华绒螯蟹的抗病过程。3酚氧化酶原激活酶(PPAF)的活化以及PPAF活化酚氧化物酶原是酚氧化物酶系统行使非特异性免疫功能的关键步骤。本研究成功克隆了中华绒螯蟹PPAF基因全长cDNA序列。该序列全长为1,386 bp,其中包括154 bp 5’-UTR,1,134bp ORF和95 bp 3’-UTR,编码378个氨基酸。该编码蛋白含有信号区域、半胱氨酸折叠区域和保守的丝氨酸区域。该。PPAF氨基酸序列与其它无脊椎动物PPAF氨基酸序列相比,具有很高的相似性(55%-75%)。PCR结果分析表明肝胰腺、肌肉、肠道、鳃中均有PPAF的表达。嗜水气单胞菌急性感染后6 h、12 h、48 h,与对照组相比PPAF基因mRNA表达水平均显著增加(P＜0.05)。结果表明PPAF与机体对外来病源的防御作用密切相关。4作为抗氧化酶类,超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GPx)、和谷胱甘肽-S-转移酶(GST)在清除体内过多的氧自由基、保护细胞免受氧化损伤等方面起非常重要的作用。本研究在EST序列的基础上,通过RACE技术获取了这三种酶基因全长cDNA序列。其中,SOD基因cDNA全长为1,339 bp,包括20 bp 5’-UTR,867 bp ORF和452 bp 3’-UTR,共编码288个氨基酸,没有信号肽序列,但含有Mn-SOD的标记保守序列(DVWHHAYY),说明该SOD属于Mn-SOD。序列比对发现,中华绒螯蟹Mn-SOD与凡纳滨对虾(L.vannamei)、班节对虾(Penaeus monodon)、蓝蟹(Callinectes sapidus)和罗氏沼虾(Macrobrachium rosenbergii)、克氏原螯虾(Procambarus clarkia)的Mn-SOD相似性分别为90%、89%、87%、84%和81%。SOD基因在各组织均有表达,其中在血细胞中表达量最高,而其他组织中表达量相对比较低,结果提示中华绒螯蟹SOD基因主要在血细胞中表达。以嗜水气单胞菌急性感染中华绒螯蟹,血细胞SOD基因mRNA的表达在感染后1.5 h达到最高,之后开始下降并在12 h达到最低点,12 h后表达量又开始升高,至48 h时表达量同1.5 h的表达量基本相同。但是1.5 h、12 h、48 h感染组的表达量与对照组之间SOD基因mRNA的表达水平不存在显著性差异(P＞0.05)。GPx基因cDNA全长为1,101 bp,其中包括94 bp 5’-UTR,660 bp ORF和347bp 3’-UTR。该ORF编码219个氨基酸,没有信号肽序列。ORF中没有发现编码硒半胱氨酸的序列(TGA),说明该GPx属于不含Se的GPx。该序列含有典型的过氧化物结构域,烷基过氧化还原蛋白(AhpC)结构域,硫氧还蛋白折叠结构域。序列比对发现,该基因氨基酸序列与锯缘青蟹(Scylla serrata)、地中海海绵(Suberites domuncula)和繁茂膜海绵(Hymeniacidon perlevis)GPx氨基酸序列相似性分别为81%、67%和66%。GPx基因mRNA在各组织均有表达,其中在肝胰腺中表达量最高,而血细胞中表达量相对比较低。结果提示中华绒螯蟹的GPx基因主要在肝胰腺中表达。嗜水气单胞菌感染中华绒螯蟹不同时间后,GPx基因mRNA在血细胞中的表达呈现下调作用。在感染后1.5 h,GPx基因mRNA表达量显著性低于对照组(P＜0.05),随着感染时间的延长,感染组GPx基因mRNA表达量虽然有所提高,但仍显著性低于对照组。结果提示,病菌的感染可能抑制了中华绒螯蟹的GPx的表达。GST基因cDNA全长为958 bp,包含85 bp 5’-UTR,651 bp ORF和222 bp3’-UTR。该ORF编码216个氨基酸,其中含有一个由17个氨基酸组成的信号肽。序列比对发现,该蛋白氨基酸序列与赤拟谷盗(Tribolium castaneum)和致倦库纹(Culex quinquefasciatus)GST相似性为53%、与大劣按蚊(Anopheles dirus)和库蠓(Culicoides variipennis)GST相似性为52%、与疟蚊(Anopheles gambiae)GST相似性为51%。该蛋白氨基酸序列含有一个82个氨基酸组成的G-位点和126个氨基酸组成的H-位点,另外也含有一个蛋白酶磷酸化位点和一个N-连接的糖基化位点。相似性和系统发生分析表明该GST属于delta-GST。通过实时定量PCR分析发现,中华绒螯蟹GST在肝胰腺,肌肉和血细胞中均有表达,而在鳃、胃和肠中未检测到其表达。其中在血细胞中的表达量最高,说明血细胞为其主要的合成部位。中华绒螯蟹受嗜水汽单胞菌感染后,GST在血细胞中的表达量在感染后6 h达到最大,且与对照组存在显著性差异(P＜0.05),而后表现为一个下降趋势,在感染12 h后恢复到起始水平。结果表明,GST基因不仅在解毒方面起重要作用,同时还参与中华绒螯蟹的免疫反应。更多还原

【Abstract】 The freshwater Chinese mitten crab,Eriocheir sinensis,is an economically important species cultured in China in the last few decades.With the development of intensive aquaculture,various diseases caused by bacteria,viruses and rickettsia-like organisms have emerged,and started to threaten the sustainability of the aquacultured populations of the mitten crab,severely affecting its production.In the present study, we presented the methodology and results of EST analysis,targeting immune-related genes in the Chinese mitten crab and conducted experiments to further clone the immune-related gene by reverse transcript-polymerase chain reaction(RT-PCR)and rapid amplification of cDNA ends(RACE)methods from haemocytes of Chinese mitten crab,compare its sequence with other related proteins.Based on the expression of the immune-related gene in various tissues,we evaluated the immune response by Real-time quantitative reverse transcription-PCR(qRT-PCR)when the E.sinensis were injected with a gram-negative bacterium,Aeromonas hydrophila.The identification and isolation of immune-related genes in crab will improve our understanding of crustacean immunity and lead to the development of new therapeutics or bacterin.1 To identify distinctive genes associated with immunity in the Chinese mitten crab(E.sinensis),an expressed sequence tag(EST)library was constructed from haemocytes of this economically important species.3,118 clones were unidirectionally sequenced and analyzed by homology searches against sequences in the GenBank,KEGG and Uniprot.Significant homology to known genes was found in 488 of the 1,049 unique sequences.The automatic functional classification based on KEGG and Gene Ontology revealed 26 putative immune-related genes.These genes coded for enzymes and proteins in the clotting and prophenoloxidase-activating system,antioxidative enzymes,antimicrobial peptides,and pattern recognition molecules.The existence of these molecular processes in the activation of cellular defense in crab has not been previously reported.According to EST abundance,the major immune-related genes were the Kazal-type proteinase inhibitor and the C-type lectin.The EST sequences of mitten crab haemocytes provide important information for understanding the evolution of the immune system among crustaceans in general.2 The lipopolysaccharide andβ-1,3-glucan binding protein(LGBP)is a pattern recognition protein which is fundamental for the innate immune response of crustaceans.The full-length cDNA of LGBP gene cloned from E.sinensis LGBP gene was 1,236 bases long and was capable of encoding a polypeptide of 362 amino acids showing significant similarity to homologous genes in shrimp.The crab LGBP deduced amino acid sequence carrying conserved features of this gene family including a potential recognition motif forβ-1,3 linkages of polysaccharides and putative RGD(Arg-Gly-Asp)cell adhesion sites.LGBP gene mRNA expression was detected in haemocytes,hepatopancreas,muscles,gills,stomachs,and intestines with the highest level in haemocytes and the lowest in the stomach.The LGBP gene expression is up-regulated upon bacterial infection and the binding of lipopolysaccharide andβ-1,3-glucan to LGBP could induce a series of immune reactions.The temporal expression of the LGBP gene after having bacterial challenge was up-regulated at 1.5 h post-injection of bacteria followed by a step by step recovery at 12 and 24 h.Our data suggest that the crab LGBP is an inducible acute-phase protein that could be critical in crab immunity.3 The proteolytic activation of prophenoloxidase(proPO)played a critical role of resisting pathogen invading in crustaceans.Analysis of the nucleotide sequence of E.sinensis PPAF genes c DNA revealed that the PPAF gene cDNA consists of 1386 bp with an open reading frame(ORF)of 1,134 bp,a 154 bp 5’-untranslated region, and a 95 bp 3’-untranslated region containing a poly A signal.The open reading frame encoded a protein of 378 amino acids with 16 residues signal sequence.The E. sinensis PPAF amino acids sequence contained putative functional domains including a signal peptide region,cysteine residues forming the clip domain and conserved serine proteinase domain.Alignment of the deduced amino acid sequence to other species PPAF revealed that PPAF was highly similar to other PPAF from crustaceans with identities from 70%to 55%.The conserved domains and clip domains,and higher similarity with other PPAF or prophenoloxidase-activating protein(PAP) suggested that PPAF was a member of the serine proteinase homgones family.The mRNA transcripts of PPAF could be detected in all the examined tissues with the highest level in hepatopancreas and PPAF transcripts in haemocytes of E.sinensis increased significantly in 6,12 and 48 h post-Aeromonas hydrophila injection.4 In order to protect against oxidative stress,aerobic cells regulate excessive ROS by a group of antioxidant enzymes,such as superoxide dismutase(SOD), glutathione peroxidase(GPx),glutathione-S-transferase(GST).These three antioxidant enzymes were all cloned from the haemocytes of Chinese mitten crab.The full-length cDNA of SOD genes consists of 1,339 bp with a 867 bp open reading frame,encoding 288 amino acids.The deduced amino acid sequence contains a putative signature sequence of Mn-SOD(DVWHHAYY).Sequence comparison showed that the cMn-SOD of E.sinensis shares 90%、89%、87°%、84%and 81% identity with that of Litopenaeus vannamei、Penaeus monodon、Callinectes sapidus、Macrobrachium rosenbergii and Procambarus clarkia,respectively. cMn-SOD transcripts were detected in all the examined tissues with the highest level in haemocytes and the temporal expression of the SOD gene after bacterial challenge was up-regulated at 1.5 h post-injection of bacteria followed by a step by step recovery at 12 h,and increased the level same as the 1.5 h at 48 h post bacterial injection.cDNA encoding glutathione peroxidase(GPx)mRNA of E.sinensis was obtained from haemocytes.The l,101bp cDNA contained an open reading frame of 660 bp,a 94 bp 5’-untranslated region,and a 347 bp 3’-untranslated region containing the poly A tail.It contains no putative selenocysteine residue which is encoded by the unusual stop codon,TGA.The conserved GPx domain,AhpC domain and the signature of peroxidase catalytic center were also identified in the crab GPx.Comparison of the crab GPx gene amino acid sequences showed similarity of 81%to that of Scylla serrata、67%to that of Suberites domuncula and 66%to that of Hymeniacidon perlevis.The mRNA transcript of GPx could be detected in all the examined tissues with highest expression level in hepatopancreas.The expression level of GPx in haemocytes was down-regulated after bacterial challenge.As time progressed,the expression level began to increase but did not recover to the original level during the experiment.The full-length GST gene cDNA comprised of 958 bp,containing 85 bp in the 5’-UTR,651 bp in the ORF,222 bp in 3’-UTR with a poly(A)tail of 25 bp and a putative polyadenylation consensus signal(AATAAA)seventeen nucleotides upstream the polyA tail.The ORF encodes a polypeptide of 216 amino acids including a 17 amino acid signal peptide.Sequence comparison of the GST deduced amino acid showed similarity of 53%to that of Tribolium castaneum and Culex quinquefasciatus GST,52%to that of Anopheles dims and Culicoides variipennis GST,51%to that of Anopheles gambiae and Anopheles dirus GST.We also discovered that GST had a G-site(1-82 aa),which binds the tripeptide glutathione in the N-terminal region and an H-site(88-213 aa),which is a substrate binding site in the C-terminus.Additionally,a kinase C phosphorylation site(ITI)and one putative N-linked glycosylation sites(DNIT)for N-linked carbohydrate chains were identified suggesting that the GST is a glycoprotein.The mRNAs of GST was detected in haemocyte,hepatopancreas and muscle with different levels of expression while was not detected in gill,intestis and stomatch.mRNAs of the gene showed the highest expression levels in haemocytes.The temporal expression of the GST gene in the control and bacteria-challenged samples is shown that the expression of GST was up-regulated significantly(p＜0.05)at 6 h post-injection of bacteria,followed by a step by step recovery at 12 h.These results suggested antioxidant enzymes genes were involved in the response to bacterial infection and played functional importance role in the immune system of E.sinensis.更多还原