中华绒螯蟹（Eriocheir sinensis）cDNA文库的构建、EST分析及其酚氧化酶系统关键基因的研究

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	中华绒螯蟹（Eriocheir sinensis）cDNA文库的构建、EST分析及其酚氧化酶系统关键基因的研究
其他题名	The cDNA library construction, expressed sequence tags (ESTs) and prophenoloxidase system of Chinese mitten crab Eriocheir sinensis
	盖云超
学位类型	博士
	2009-05-28
学位授予单位	中国科学院海洋研究所
学位授予地点	海洋研究所
关键词	中华绒螯蟹 Dna文库表达序列标签（est）固有免疫鳗弧菌酚氧化酶实时定量pcr Rnai 酚氧化酶激活酶丝氨酸蛋白酶抑制剂调控机制
摘要	中华绒螯蟹（Eriocheir sinensis）是我国的特色物种，具有重要的经济和科研价值。酚氧化酶系统作为节肢动物特有的免疫机制，在中华绒螯蟹的免疫反应中发挥重要作用。本研究构建了一个中华绒螯蟹的cDNA文库，利用表达序列标签 (Expressed Sequence Tag，EST) 技术，对中华绒螯蟹表达序列进行了大规模测序分析，并利用cDNA末端快速扩增（rapid amplification of cDNA ends，RACE）、实时定量PCR、原核重组和RNAi等技术研究了其酚氧化酶免疫系统的分子基础及其相应功能。用鳗弧菌和金黄色葡萄球菌同时感染中华绒螯蟹，提取血细胞的RNA构建了一个库容为3.3×106 克隆cDNA文库。随机测序后获得7535条高质量的EST序列，其中在GenBank数据库中未发现同源序列的为4593 条，而具有较高同源性2942条可以分为20个功能类别，参与了23个生物学反应。进一步分析发现，969 条（32.9% ）EST与免疫相关，可拼接成221个免疫基因。这个比例高于其它任何一个已公布的甲壳动物cDNA文库。在免疫相关EST中，抗菌肽比例最高，约占总数的20.1%（195条EST）。免疫基因的高比例和抗菌肽的高表达，证明细菌刺激是提高cDNA 文库中免疫基因丰度的有效方法。 EST序列的获得和免疫基因的富集，丰富了中华绒螯蟹的基因组信息，初步了解了中华绒螯蟹固有免疫系统的概况, 为进一步克隆和研究中华绒螯蟹免疫防御功能基因提供了序列基础。本研究在EST分析的基础上，克隆获得了中华绒螯蟹酚氧化酶系统10个基因的cDNA全长序列, 它们分别是前酚氧化酶（EsproPO），丝氨酸蛋白酶同源物（EsSPH), 丝氨酸蛋白酶抑制剂pacifastin, serpin, PAPII (EsPLC, Es serpin, EsPAPII), 模式识别丝氨酸蛋白酶（EsPRSP），peroxinectin (Esperoxinectin)和3个前酚氧化酶激活酶 (EsPAP1, 2, 3)。它们与相近物种的酚氧化酶系统相应基因均具有较高同源性，并含有胰酶催化结构域，CLIP结构域，PLD结构域，KAZAL结构域，Serpin结构域以及酚氧化酶结构域等酚氧化酶系统相应基因典型的特征结构域。分析发现，PAPs的CLIP结构域和PRSP，Pacifastin，Proxinectin，proPO基因是节肢动物特有的，是酚氧化酶系统作为节肢动物特有免疫机制的分子基础。本研究从多个基因的3′UTR区发现了调控元件，如15-LOX-DICE，K-box和 Brd-Box。在所推断的蛋白中，EsPAP3和EsPAPII的等电点呈碱性，Esperoxinetin的为中性，而EsPRSP，EsSPH，EsproPO, EsPAPII, Esserpin，EsPAP1的等电点在酸性区间。健康中华绒螯蟹 EsPAP1，EsPAP2，EsPAPII基因在肌肉中的表达量最高，而在血细胞中的表达量相对较低；EsPAP3，EsproPO，EsPLC基因在血细胞中表达量较高，在肌肉中的表达量最低。其中，EsPAP3在血细胞中的表达量是其在肌肉组织中表达量的526.35倍。调控元件和多种激活酶与抑制剂的存在、组织分布和等电点的差异，说明中华绒螯蟹酚氧化酶系统在转录、翻译、激活等多个层次上受到了调控。在中华绒螯蟹受到鳗弧菌刺激后，EsPAP1，EsPAP2，EsPAP3，EsPLC和EsPAPII基因的表达量呈上升或下降的趋势，但表达量的极限值均出现在2小时和12小时，这一规律与EsproPO应激后的mRNA表达和酶比活力的变化特点相吻合，说明中华绒螯蟹酚氧化酶系统各因子相互协调共同参与中华绒螯蟹对入侵细菌的防御反应。同时EsPAP2，EsPAP3，EsproPO，EsPAPII，EsPLC在中华绒螯蟹受到鳗弧菌刺激后的表达呈现反复多次上升，表明酚氧化酶系统可能参与了多种免疫反应。研究还发现EsPAP1参与中华绒螯蟹血液凝集过程，而EsPAP3是蟹血细胞中的有效的前酚氧化酶激活因子。研究结果初步揭示了中华绒螯蟹酚氧化酶系统的分子基础、对微生物的响应机制及其调控机制和演化趋势，为节肢动物固有免疫系统研究奠定了良好基础。
其他摘要	Chinese mitten crab Eriocheir sinensis (Henri Milne Edwards 1854) is one important aquaculture species in China, possessing striking economic and scientific value. The prophenoloxidase activating system (proPO system), playing key roles in the immune defense response of Chinese mitten crab, is the unique immune component of arthropod innate immunity. In the present study, a cDNA library was constructed and sequenced. The resultant ESTs (Expressed Sequence Tags) were analyzed with bioinformatics tools. RACE (rapid amplification of cDNA ends), real time RT-PCR, protein recombinant and RNAi techeniques were adopted to investigate the cDNA characteristic, mRNA expression and molecular function of key genes of E. sinensis proPO system. In the present study, a high quality cDNA library of Chinese mitten crab haemocytes challenged by bateria was constructed with its capacity up to 3.3×106. After random sequencing, 7535 ESTs were obtained and submitted to the GenBank database. Among them, 4593 ESTs having no significant matches to any protein sequences in the public database were identified as novel genes, and the other 2942 were identified as matched genes. Based on the Gene Ontology (GO) classification system, matched genes were classed into 20 GO classifications in terms of molecular functions or 23 GO groups in terms of biological processes. After EST analysis, 32.9% (969 out of 2942) ESTs were identified as immune genes, which were assembled into 221 unigenes. This percentage was significant higher than that of previous reported crustacean cDNA libraries. In addition, antimicrobial peptides (AMPs) taken up 20.1% (195 ESTs) of the 969 ESTs. The high percentage of immune genes 7535 ESTs and the high expression of AMPs indicated that bacteria stimulation was a useful method for improving the proportion of immune genes in cDNA library. The results mentioned above greatly enriched the genomic information of Chinese mitten crab, laid the first step for the elucidation of crab immune system, and for the first time made the full-scale study of the molecular mechanism under various biological processes of crab possible. In the present study, interests were focused on the molecular characteristic, tissue distribution, temproral expression profiles of ten proPO system members of Chinese mitten crab as well as the regulation machnism and evolution trend of E. sinensis proPO system. Ten full-length cDNAs were obtained from E. sinensis based on EST analysis and RACE (rapid amplification of cDNA ends) technique, including the PRSP (EsPRSP), PAP1 (EsPAP1), PAP2 (EsPAP2), PAP3 (EsPAP3), SPH (EsSPH), proPO (EsproPO), PAPII (EsPAPII), serpin (Esserpin), pacifastin (EsPLC), peroxinectin (Esperoxinectin). Blast analysis and multiple sequence alignment indicated that all these genes were members of the proPO system. Furthe study indicated that the CLIP domain of PAPs, the genes of PRSP, pacifastin, proxinectin and proPO could only be identified in arthropod, which might be the molecular basis for the uniqueness of the proPO system in arthropod. Some regulation elements, such as 15-LOX-DICE, K-box and Brd-box, were identified in the 3′UTR of proPO system genes. The PIs of crab proPO system factors presented significant differences. The mRNA transcripts of these genes were detected in the haemocytes, heart, gill, gonad, muscle, and hepatopancreas. The mRNA of EsPAP1, EsPAP2, EsPAPII were predominately expressed in muscle and lower expressed in haemocytes; Mealwhile, the expression of EsPAP3, EsproPO and EsPLC was highest in haemocytes and lower in muscle. It was notable that the expression level of EsPAP3 in haemocytes was 526.35-fold to that in muscle. The existence of regulation elements in the 3′UTR of proPO system genes and the presentce of more than one proPO activating proteases as well as protease inhibitors, the difference of PIs and mRNAs expression in tissue distribution collectively implied that the proPO system of E. sinensis was regulated at least on the transcription, translation, enzyme activation, and signal pathway levels. The temporal expression profiles of proPO system members in the crabs challenged with Listonella anguillarum were obtained with the Realtime PCR technique. Though some genes was found increase the mRNA expression while the others decrease, the expression levels of EsPAP1, EsPAP2, EsPAP3, EsPLC and EsPAPII reached the extremum at same schedule (2h, 12h). This expression characteristic was similar to that of the mRNA expression and specific activity of EsproPO after L. anguillarum challenge, which implied that EsPAP1, EsPAP2, EsPAP3, EsPLC, EsPAPII might cooperate together with EsproPO to play roles in the immune process of Chinese mitten crab. In addition, the expression of EsPAP2, EsPAP3, EsproPO, EsPAPII and EsPLC increased twice after the bacteria challenge. This multiple up-regulation revealed that the proPO system probably functioned in several immune processes during the interaction between the host and bacteria. Using RNAi method, EsPAP3 was further proved to be an effective proPO activating factor in haemocytes, and EsPAP1 played roles in the coagulation of Chinese mitten crab. Taken together, the present study identified the molecular basis for the uniqueness of the proPO system in arthropod, obtained the mRNA expression characteristic of proPO system members of Chinese mitten crab and provided new insights into the regulation machnism of E. sinensis proPO system.
页数	196
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/289
专题	海洋环流与波动重点实验室
推荐引用方式 GB/T 7714	盖云超. 中华绒螯蟹（Eriocheir sinensis）cDNA文库的构建、EST分析及其酚氧化酶系统关键基因的研究[D]. 海洋研究所. 中国科学院海洋研究所,2009.

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