【作者】 蒋惠；

【导师】 王群；

【作者基本信息】 华东师范大学 ， 水生生物学， 2011， 博士

【摘要】 中华绒螯蟹（Eriocheir sinensis）是中国淡水渔业的重要组成部分,它已经成为中国的一道传统美食。由于它肉质细嫩,味道鲜美,营养丰富,在全球市场供不应求且价格不菲。它是我国重要的经济水产养殖品种,该蟹的养殖已成为我国水产养殖的支柱产业。在过去的几十年中,随着密集化养殖的发展,由细菌、病毒和类立克次氏体属微生物所引发的各种疾病,以及性早熟、个体小型化以、种质退化等现象在中华绒螯蟹种群中频频发生,并且对螃蟹的质量造成灾难性损失,已经成为限制该蟹养殖业发展的重要瓶颈。因此认为了解其免疫防御机制、免疫因子特征,以及生殖生物学尤其是生殖的分子机理的研究,对螃蟹养殖的疾病控制和健康管理很有帮助,并为该蟹养殖过程中的人工生殖调控提供理论保障。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及雄性生殖机理,以期能有效突破中华绒螯蟹人工养殖的瓶颈,进一步促进该蟹养殖业的持续发展。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肝胰腺cDNA文库的构建和表达序列标签（EST）的分析,并利用先进的Solexa测序技术,对中华绒螯蟹副性腺组织进行转录组及microRNA的大规模测序并分析其基因表达情况。不仅丰富了中华绒螯蟹的基因组数据库,同时筛选出许多免疫相关及生殖相关的的基因序列。通过对所测高通量数据分析的注释结果,结合已有物种生殖相关基因的研究现状,分析、筛选与该蟹生殖发育相关的部分重要基因；利用RACE和WORKING等技术,获得这些基因的cDNA全长以及基因的结构；利用realtime PCR研究不同基因在不同组织中以及同一组织不同发育阶段的表达情况,探讨这些基因在整个生殖发育过程中的调控作用。这些研究为了解中华绒螯蟹生殖机制提供了基础资料,同时也为深入探讨该蟹雄性生殖机理提供了重要的参考。主要研究结果如下：1采用常规cDNA文库构建技术,成功构建了一个中华绒螯蟹肝胰腺的EST文库并且获得了3,279高质量序列,代表着1177个一致性序列。超过一半的一致性序列被鉴定是这一物种的未知的新基因。有很大一部分一致性序列（49%）在新发布的数据库中找到它们的同源物,这些同源物对于进一步的功能研究很重要。总结了与代谢和免疫相关的基因,借以考察肝胰腺的两种重要功能：代谢和免疫。为中华绒螯蟹的深入研究打下基础,包括转录组、物理图谱和整个基因组测序。展示了中华绒螯蟹与免疫密切相关的转录组和基因的重要性质。本文重点找到了与机体免疫相关的基因,分为5大类,包括蛋白酶类、抗菌肽、压力蛋白、抗氧化蛋白和其它与免疫相关的基因进行重点分析。蛋白酶类和他们的抑制剂在无脊椎动物抗感染反应中的重要性越来越明显。它们可能在应对侵染性蛋白的体液免役反应过程中表达,这些侵染性蛋白是由侵染性抗原产生的。文库中发现了1种抗菌肽,抗脂多糖因子。压力蛋白包括铁蛋白、金属硫蛋白、热激蛋白70、胞溶质蛋白A。硒与谷胱甘肽系统在抗氧化防御体系。以及p-1,3-葡聚糖结合蛋白、C型凝集素、血蓝蛋白、干扰素等,都对中华绒螯蟹的免疫有着重要作用。2从雄性中华绒螯蟹的副性腺组织中提取并分离mRNA,经超声破碎和随机引物反转成cDNA片段,利用Solexa测序（sequencing by synthesis）技术,对这些组织的RNA转录组进行全面测序；用生物信息学的手段和方法,对测序数据进行转录谱分析,分析各组织的基因表达情况,进一步综合各组织之间基因表达的相关性,探讨不同组织在该蟹雄性生殖过程中的作用及调控模式。共获得33,221,284条reads,包含2,657,702,720nt碱基,GC含量55.19%。通过软件组装拼接得到85,913条contig,将所有contig按序列长度从小到大排序,其中40,977条（47.70%） contig集中在100-200nt的长度.从第一条序列开始累加计算contig总长,所有contig的总长度为18,750,722。通过对Unigene的功能注释,其中有1713条基因条注释到General function prediction only,注释上Translation, ribosomal structure and biogenesis有952条基因,与Transcription相关的有869条Unigene。关于Posttranslational modification, protein turnover, chaperones和Replication, recombination and repair的基因分别有802和789条。KEGG是基因组破译方面的数据库。附性腺有1704条基因（14.99%）参与Metabolic pathways,675条基因参与Regulation of actin cytoskeleton。Spliceosome有564（4.96%）条基因参与其中。得到原始小RNA的fq数据后,对其进行去接头,去低质量reads,去污染等处理,得到干净的序列7939380条。统计小RNA（sRNA）的种类（用unique表示）及数量（用total表示）,并对小RNA做长度分布统计,一般来说,小RNA的长度区间为18-30nt,通过长度分布的峰我们可以判断小RNA的种类,miRNA集中在21或22nt, siRNA集中在24nt, piRNA集中在30nt。共有Total sRNAs 7939380条,其中miRNA有382650条（4.82%）、rRNA有214013条（2.70%）、snRNA有2573条（0.03%）、snoRNA有563条（0.01%）、rRNA有432171条（5.44%）以及unann有6907410条（87.00%）。共有Unique sRNAs 2268484条,其中miRNA有23021条（1.01%）、rRNA有33764条（1.49%）、snRNA有1003条（0.04%）、snoRNA有224条（0.01%）、tRNA有29425条（1.30%）和unann有2181047条（96.15%）。3生殖相关蛋白及其功能的研究是当前甲壳动物生殖生物学的研究重点之一,许多生殖相关蛋白及其功能已经明确。瘦素受体在生殖发育过程有调节生殖发育的作用。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瘦素受体蛋白（EsLEPR）cDNA全长。生物信息学分析发现,该基因cDNA全长序列的长度大小1403 bp包括一个72 bp的5’非翻译区（5’ UTR）,1929 bp的3’-非翻译区（3’UTR）的一个AATAAA加尾信号和23 bp的polyA尾,和402 bp的开放阅读框（ORF）,编码133个氨基酸的多肽与预测分子量14.42kDa和理论等电点为4.46。其基因组DNA序列为3553bp其中有3个内含子和4个外显子组成,包含有典型的（GT和AG）二核苷酸内含子交界处的结构序列剪接位点。氨基酸序列中有一个保守区域：Vacuolar protein sorting 55 （Vps55）。133个氨基酸的序列中的保守区域含有4个跨膜束片段,分别在第4到第26个氨基酸,第33到第52,第67到第89,和第101到第123。氨基酸序列的多重比对结果显示与Eriocheir sinensis同源性最高的是（64%）丽蝇蛹集金小蜂（Nasonia vitripennis）。它与非洲爪蛙（Xenopus laevis,63%）、红鳍东方触（Takifugu rubripes,58%）、人（Homo sapiens,57%）、牛（Bos taurus,57%）、（Rhodnius prolixu,56%）、褐鼠（Rattus norvegicus,55%）、海鞘（Ciona intestinalis,54%）,、猕猴（Macaca mulatta,54%）,、蚜虫（Acyrthosiphon pisum,48%）和（Boltenia villosa,40%）都有较高的同源性。采用实时定量RT-PCR的方法分析中华绒螯蟹瘦素受体蛋白在不同组织中以(3-actin作为内参的相对表达情况。组织中的表达分析显示,中华绒螯蟹瘦素受体蛋白的mRNA在各个组织中均有表达,主要是在肠、精巢、胸神经节、脑神经节和肝胰腺等组织中表达。4蜕皮激素引起的幼体蜕皮发育有关的各种活动。蜕皮激素调节蛋白可能是甲壳动物变态蜕皮级联反应的重要元素。在中华绒螯蟹养殖中性早熟和蜕皮未遂的问题经常发生。这两种病害与甲壳动物的蜕皮是密切相关的,研究蜕皮相关基因对于解决中华绒螯蟹性早熟和蜕皮未遂的问题有重要的意义。通过EST分析得到蜕皮激素调节蛋白的两条ESTs （FG358768,FG359773）；用RACE方法得到中华绒螯蟹蜕皮激素调节蛋白cDNA全长。中华绒螯蟹蜕皮激素调节蛋白基因cDNA全长序列的长度大小1384 bp包括一个117-bp的5’非翻译区（5’UTR）、841-bp的3’-非翻译区（3’UTR）的一个AATAAA加尾信号和31-bp的polyA尾,和426 bp的开放阅读框（ORF）,编码141个氨基酸的多肽与预测分子量15,150.3 Da和理论等电点为3.86。中华绒螯蟹蜕皮激素调节蛋白的基因组DNA序列为3060bp其中有1个内含子和2个外显子组成。中华绒螯蟹蜕皮激素受体基因组序列的包含有典型的（GT和AG）二核苷酸内含子交界处的结构序列剪接位点。而且在基因组序列序列与其cDNA序列一致。用ClustalW软件对不同物种的蜕皮激素调节蛋白的氨基酸序列进行比对。中华绒螯蟹的蜕皮激素调节蛋白与Litopenaeus vannamei的蜕皮激素调节蛋白（ABD65303）同源性为34.4%,与Bombyx mori蜕皮激素调节蛋白（NP₀01134647）的同源性为25.8%。序列比对结果显示有6个高度保守的半胱氨酸残基（Cys22, Cys36, Cys41, Cys87, Cys92, Cys133）。SMART程序分析表明,推导的氨基酸序列包含一个保守的结构域:the Niemann-Pick type C2（属于ML家族）结构域。是由119个氨基酸组成开始在Tyr19并在Val138结束。在ML保守结构域上有22个公认的胆固醇/脂质结合位点。采用SWISS-MODEL模型预测中华绒螯蟹蜕皮激素调节蛋白的三级结构,根据与其他物种相同蛋白很高的相似性,建立的三级结构的模板为:lnepA。模拟结构的氨基酸残基范围开始于第16个残疾和结束于第141残基和序列结构同源性为28.9%。在整个中华绒螯蟹蜕皮激素调节蛋白推导的氨基酸序列结构主要是β-折叠。采用实时定量RT-PCR的方法分析中华绒螯蟹蜕皮激素调节蛋白在不同组织中以β-actin作为内参的相对表达情况。组织中的表达分析显示,中华绒螯蟹蜕皮激素调节蛋白的mRNA主要是在肝胰腺组织中表达,在脑神经节中表达相对较高,在性腺、胃、肌肉和胸神经节中表达较少,而在心,鳃,血淋巴和肠中不表达。在肝胰腺中的表达最丰富,这与肝胰腺的功能密切相关,肝胰腺是蜕皮现象的主要指示器官。采用实时定量RT-PCR的方法,分析中华绒螯蟹蜕皮激素调节蛋白在中华绒螯蟹不同蜕皮时期（蜕皮前、蜕皮中、蜕皮后和硬壳期）肝胰腺中的表达情况。中华绒螯蟹蜕皮激素调节蛋白在蜕皮前的肝胰腺中表达量最高,在进入蜕皮时显著降低,蜕皮后开始回升,直到硬皮期回升到正常水平。更多还原

【Abstract】 The mitten crab (Eriocheir sinensis) (Henri Milne Edwards 1854), which belongs to Crustacea, Decapoda, Grapsidae, Eriocheir, is a unique species of crab that is native to China. This species is a traditional savory food and therefore comprises one of the economical aquatic species of China. The farming of E. sinensis has been developing rapidly in China over the last two decades and reached its highest yield of 4.0 x 105 tonnes in 2005. With the development of intensive culture, various diseases caused by bacteria, viruses, or rickettsia-like organisms and the sexual precocity, individual small, germplasm degradation are frequent in cultured species populations and have caused catastrophic losses. It has become a major bottleneck in the development of aquaculture industry. Understanding the immune defence mechanisms of the species; characterizing of immune effectors, and the study of reproductive biology, especially the molecular mechanism of reproductive are believed to be helpful in health management and disease control in crab aquaculture, and provide a theoretical to the reproductive control of the artificial crab breeding process.In the present study, we presented the methodology and results of transcriptome analysis, targeting immune-related genes and reproduction-related genes in the Chinese mitten crab and conducted experiments to further clone the reproduction-related gene by reverse transcript-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods from Chinese mitten crab, compare its sequence with other related proteins. We evaluated the expression of the reproduction-related gene in various tissues by Real-time quantitative reverse transcription-PCR (qRT-PCR), to explore the regulation of reproductive function of these genes during the development. The study is to provide a basis for Chinese mitten crab reproductive mechanisms information, but also provide an important reference for further research on the mechanism of the male reproductive in Chinese mitten crabs. The main results are as follows:1 Due to its popularity as a traditional food, intensive harvesting of the mitten crab (Eriocheir sinensis) is common, and has lead to an increase in disease incidence, resulting in catastrophic losses to crab aquaculture. The hepatopancreas of E. sinensis is not only an important digestive organ but also an indispensable immune organ. We constructed a non-normalized cDNA library from the hepatopancreas of E. sinensis and acquired 3,297 high-quality expressed sequence tags representing 1,178 unigenes. More than half of these unigenes were novel genes for this species; the remaining had homologs in public databases, which is of great importance for future functional research. We also investigated the association of these genes with immune processes for insight into one of the main functions of the hepatopancreas besides metabolism. Despite the relatively low sampling scalar of our cDNA library, we were able to demonstrate several important properties of the hepatopancreatic transcriptome and identified numerous genes that were closely associated with immune responses. These results might serve as the basis for an in-depth genomics study of E. sinensis, including transcriptome analysis, physical mapping, and whole genome sequencing.2 Beads with Oligo(dT) are used to isolate poly(A) mRNA after total RNA is collected from accessory sex gland of the Chinese mitten crab. Fragmentation buffer is added for interrupting mRNA to short fragments. Taking these short fragments as templates, random hexamer-primer is used to synthesize the first-strand cDNA. The second-strand cDNA is synthesized using buffer, dNTPs, RNaseH and DNA polymerase I, respectively. Short fragments are purified with QiaQuick PCR extraction kit and resolved with EB buffer for end reparation and adding poly(A). After that, the short fragments are connected with sequencing adapters. And, after the agarose gel electrophoresis, the suitable fragments are selected for the PCR amplification as templates. At last, the library could be sequenced using Illumina HiSeqTM 2000. Transcriptome de novo assembly is carried out with short reads assembling program-SOAPdenovo. A total of 33,221,284 read including total nucleotides 2,657,702,720 nt, which generated 91.06% successful reads. Of these, the average G+C content was 55.19%. These reads were assembled by software consisting of 85,913 contigs, of which 40,977 (47.70%) contigs with the mass of lengths ranging from 100-200nt length. The total length of all the contig is 18,750,722. Functional annotation by Unigene, of which 1713 genes annotated to Article General function prediction only, comment on the Translation, ribosomal structure and biogenesis were 952 genes, associated with the Transcription has 869 Unigene. About Posttranslational modification, protein turnover, chaperones, and Replication, recombination and repair genes were 802 and 789. KEGG is deciphering the genome’s database. The accessory sex gland of Chinese mitten crab with 1704 genes (14.99%) in Metabolic pathways, 675 genes involved in Regulation of actin cytoskeleton. Spliceosome has 564 (4.96%) genes involved. Get the original small RNA of fq data, to its connection to low-quality reads, to deal with pollution, access to clean sequence 7939380. Statistics of small RNA (sRNA) species and quantity, and the length distribution of small RNA to do statistics, in general, the length of the small RNA interval 18-30nt, through the length distribution of the peak we can determine the type of small RNA, miRNA concentrated in 21 or 22nt, siRNA concentration in the 24nt, piRNA concentrated in the 30nt. Total Total sRNAs are 7939380, of which miRNA are 382,650 (4.82%), rRNA with 214,013 (2.70%), snRNA with 2573 (0.03%), snoRNA with 563 (0.01%), tRNA with 432,171 (5.44%), unann with 6,907,410 (87.00%). Total Unique sRNAs are 2268484, of which miRNA are 23,021 (1.01%), rRNA with 33,764 (1.49%), snRNA with 1003 (0.04%), snoRNA with 224 (0.01%), tRNA with 29,425 (1.30%), unann with 2,181,047 (96.15%).3 Leptin is an adipocyte-derived hormone with multiple functions that regulates energy homeostasis and reproductive functions. Increased knowledge of leptin receptor function will enhance our understanding of the physiological roles of leptin in animals. In the present study, a full-length leptin receptor (lepr) cDNA, consisting of 1,353 nucleotides, was cloned from Chinese mitten crab(Eriocheir sinensis) using rapid amplification of cDNA ends (RACE) following the identification of a single expressed sequence tag (EST) clone in a cDNA library. The lepr cDNA consisted of a 22-nucleotide 5’-untranslated region (5’UTR), a 402-nucleotide open reading frame (ORF) and a 929-nucleotide 3’UTR. Multiple sequence alignments revealed that Chinese mitten crab lepr shared a conserved vacuolar protein sorting 55 (Vps55) domain with other species. Chinese mitten crab lepr expression was determined in various tissues and at three different reproductive stages using quantitative real-time RT-PCR. Lepr expression was highest in the intestine, thoracic ganglia, gonad, and accessory gonad, moderate in hepatopancreas and cranial ganglia, and low in muscle, gill, heart, haemocytes, and stomach. Furthermore, lepr expression was significantly higher in the intestine, gonad and thoracic ganglia in immature crabs relative to precocious and mature crabs. In contrast, lepr expression was significantly lower in the hepatopancreas of immature crabs relative to mature crabs. We are the first to identify the lepr gene and to determine its gene expression patterns in various tissues and at three different reproductive stages in Chinese mitten crab. Taken together, our results suggest that lepr may be involved in the nutritional regulation of metabolism and reproduction in Chinese mitten crabs.4 Ecdysteroid causes various developmental events associated with larval-larval ecdysis. Ecdysteroid-regulated gene could be a putative element of the metamorphic ecdysone response cascade of crustacean. The ecdysteroid-regulated genes mRNA level correlates well with the haemolymph ecdysteroid titre during the molting process. A full-length cDNA of ecdysteroid-regulated gene from Eriocheir sinensis was cloned by the rapid amplification of cDNA ends (RACE), with primers that designed based on the EST sequence of the cDNA library of hepatopancreas from E. sinensis. The cDNA is 1384 bp in size and consists of a 117-bp 5’-untranslated region (UTR), a 306-bp open reading frame (ORF) and an 841-bp 3’-UTR. The genomic DNA is 3059-bp containing two exons interrupted by one intron. Multiple alignments revealed that a conserved Niemann-Pick type C2 domain was identified in the deduced amino acids sequence of ecdysteroid-regulated gene. The mRNA expression of ecdysteroid-regulated gene in ten tissues and the temporal expression in hepatopancreas of four phases were measured by real-time RT-PCR. Ecdysteroid-regulated gene mRNA transcripts could be detected in all examined tissues, and are higher expressed in hepatopancreas than that in other nine tissues (cranial ganglia, P<0.05; the rest eight tissues, P<0.01). Among the four phases, the gene expression at pre-molt is the highest, down-regulated during inter-molt, up-regulated at post-molt, and continues to increase in hard-shell phase. The mRNA expression pattern of ecdysteroid-regulated gene confirmed that the hepatopancreas is a sensitive indicator for ecdysis phase in various crustaceans, and ecdysteroid-regulated gene would be involved in the process of crustacean molting. Therefore, this study by E. sinensis Ecdysteroid-regulated gene cloning and expression analysis, contribute to clarify the mechanism of molting occurs, and may be helpful for the prevention of related problems.更多还原