【作者】 阮灵伟；

【导师】 徐洵；

【作者基本信息】 厦门大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 深海热液区是地球上典型的极端环境，尽管环境条件极其严酷，但以化能自养微生物为基础的自养自给的共生体系却蕴育了这里生机勃勃的生物世界。极端的环境使这里的生物形成了独特的防御与适应性机制，引发了科学家们对诸如生命起源、演化、生物耐受极端环境的机制等一系列重大生物学问题的思考；同时代谢过程中形成的各种生物活性物质也使其成为潜在的生物资源宝库。本研究以深海热液区Juan de Fuca Ridge的典型生物管状蠕虫Ridgeiapiscesae为研究对象，运用SMART技术构建了其cDNA文库，各项指标均符合文库质量标准：文库的容量为5．71×10~6个克隆，平均插入片段大小～1．0kbp。在此基础上，我们随机挑取879个文库克隆进行EST测序。序列分析表明这些序列代表199个基因，根据功能特点这些基因包括基础代谢相关蛋白、细胞骨架及膜相关蛋白、环境适应性与防御相关蛋白、细胞增殖相关蛋白、细胞信号传导相关蛋白、DNA的复制、转录及蛋白翻译相关蛋白等，比较全面的反映了管状蠕虫Ridgeia piscease的分子特征。序列分析还表明，与适应性和防御性相关的基因显示了基因的多样性。其中，几丁质结合蛋白最具多样性，不仅几丁质酶水解区和几丁质结合区相互独立，而且几丁质结合区也显示丰富的多样性，由不同的几丁质结合区共组成了23种不同的几丁质结合蛋白；EST测序分析中还发现两个溶菌酶基因带有双功能区，可能具有双重功能；此外，肌红蛋白基因也显示了序列的多样性。管状蠕虫Ridgeia picesae的这些基因特点将有助于我们了解生物对极端环境的适应性机制，同时也有利于我们开发其中潜在的基因资源。microRNA是真核生物中一类内源性的具有调控功能的非编码RNA，广泛参与生长发育、器官形成、细胞增殖与凋亡、病毒防御等各个过程。对虾作为一种重要的经济动物，对其相关microRNA的分子特征还知之甚少。本研究对日本囊对虾Marsupenaeus japonicus microRNA途径中相关的分子特征进行了初步探索。Argonaute是microRNA途径中的重要蛋白之一，microRNA通过以Argonaute为核心的RISC复合体(RNA-induced silencing complex)对目标基因进行调控。因此，我们首先从日本囊对虾中克隆得到这一关键蛋白Argonaute——Mjago，并对其分子特征进行相关研究。序列分析表明Mjago含有Argonaute蛋白家族特有的PAZ和PIWI保守结构域，并且其与节肢动物的Argonaute 1，尤其与斑节对虾Penaeus monodon的Argonaute 1具有极高的同源性。对不同组织Mjago转录水平的检测发现，该基因在对虾各个组织中均有转录，尤其以血细胞中的转录水平最高。进一步的研究发现，Mjago的转录水平与WSSV病毒感染密切相关，在病毒感染后其转录水平上升，在感染48小时后转录水平达到最高，随后转录水平又逐渐下降。在Mjago研究的基础上，我们进而开展了对日本囊对虾microRNA相关分子特征的研究。为了从免疫角度对日本囊对虾microRNA进行研究，我们以WSSV感染的血细胞为材料，从中分离并克隆小RNA。运用较为灵敏的PCR鉴定方法，从中我们鉴定得到35个microRNA。序列分析表明血细胞中部分microRNA表现出了表达丰度的差异性及进化上的保守性，其与已知物种中的microRNA具有极高的同源性，尤其是进化关系上十分接近的果蝇、家蚕等节肢动物来源的microRNA。进一步，我们运用半定量PCR方法检测所鉴定microRNA在WSSV病毒感染过程中表达水平的变化情况，结果表明部分microRNA的表达水平在病毒感染过程中发生了变化。日本囊对虾Mjago和microRNA的初步研究表明microRNA途径中的相关分子与WSSV的病毒感染存在着紧密的联系。对这些分子进行深入的研究将有助于我们认识microRNA在对虾免疫中的分子机制，为对虾的抗病毒免疫研究提供新线索，从而拓展对虾的免疫调节机制，丰富无脊椎动物的免疫学研究。更多还原

【Abstract】 Although the condition of the deep-sea hydrothermal vent is harsh,a vigorousecosystem is supported by the symbiosis between the chemoautotrophicmicroorganisms and their hostes.The creasures living there have possessed the uniquemechanism to adapt to the extreme environment.The special ecosystem is not onlypotential biological resource,but also arises the though of scientists about the lifeorigin,evolution,adaptation to the extreme environments and so on.Ridgeia piscesae,living around the hydrothermal vent of Juan de Fuca Ridge,isan ideal model for studying the adaptative mechanism to extreme environment.Forinsights of its molecular characteristics,a SMART cDNA library of 5.71×10~6 cloneswas constructed.The average length of the inserted cDNA fragments is about 1.0 kbp.A total of 879 clones were picked at random and sequenced.199 genes wereidentified for the first time.They were found to be related to basal metabolism,cytoskeleton and membrane proteins,adaptation and defense,cell proliferation,signaltransduction,DNA replication,transcription and translation and so on.Among them,we found 23 various chitin-binding proteins consist of different chitin bindingdomains and a chitinase that only contain a domain of GH18 (glycosyl hydrolasesfamily 18).Additionally,high polymorphism also exists in other genes,such asmyohemerythrin,lysozyme that maybe a muti-funtional gene.The gene-expressionprofile might help to further understand the molecular basis of tube worm physiology.It will also lay a good foundation for functional studies on the adaptation to extremeenvironments and help to make use of them. MicroRNAs (miRNAs)are a class of small noncoding RNA that are 20-22nucleotides (nts)and function as regulators of gene expression.They were extensivelystudied and it was found that they were involved in various developmental andphysiological processes,such as cell differentiation,proliferation,apoptosis,development,virus defense and so on.Shrimp is one of the most important species inthe aquaculture.However,few studies about microRNA in shrimp were performed.In this study,we present the molecular characteristics of the molecules in themicroRNA pathway from Marsupenaeus japonicus.Argonaute is one of the mostimportant components in this pathway,which constitutes the RISC (RNA-inducedsilencing complex)with other partners and guides microRNAs to regulate theexpression of the target genes.Firstly,we present the molecular characteristics of theargonaute named Mjago from M.japonicus.Sequence analysis revealed that Mjagohad the conserved domains of the argonaute family:PAZ and PIWI.And it had theclose evolutionary relationship with argonaute 1 from arthropod,especially theargonaute 1 of Penaeus monodon.As shown in RT-PCR,Mjago appeared in everytissues examined and had the highest transcriptional level in the hemocyte.Moreover,during the white spot syndrome virus (WSSV)infection,it is found that thetranscriptional level of Mjago increased rapidly,but reduced after 48 h post infection.Furthermore,the study on the molecular characteristics of microRNA from M.japonicus was performed.For investigation on their relationship to the shrimpimmunity,the WSSV infected hemocytes were collected for isolation and clone ofsmall RNA.35 microRNAs were identified by the PCR method.Sequence analysisrevealed that some microRNAs shown the differential expression and evolutionalconservation.They have high homology with the microRNAs from other organisms,especially the arthropod,such as Drosophila melanogaster,Bombyx mori and so on.The result of semi-quantitative PCR showed that the expression of some microRNA changed during the WSSV infection.The study revealed that the molecules of the microRNA pathway from M.japonicus had the close relationship with the WSSV infection.Further study on theirindividual functions will be helpful to understand the molecular mechanism ofmicroRNA in the shrimp immunity and provide a clue for researches on shrimpimmunity against virus.更多还原