【作者】 张伟；

【导师】 王群；

【作者基本信息】 华东师范大学 ， 水生生物学， 2012， 博士

【摘要】 中华绒螯蟹(Eriocheir sinensis).又称河蟹,属于甲壳纲(Crustacea)十足目(Decapoda),是我国重要的水产养殖经济品种,养殖规模逐渐的扩大。近年来,随着集约化程度的提高,出现的性早熟现象导致了成品蟹的品质严重的下降,制约了制约了中华绒螯蟹的养殖业的发展,导致经济产值下降。因此,本论文试图从分子生物学的角度初步探讨究中华绒螯蟹的生殖调控机制,为将来从分子水平解决性早熟提供坚实的理论基础和基因组资料。本研究利用分子生物学、生物信息学的技术手段,首次构建了中华绒螯蟹精巢组织cDNA文库的构建,并对得到的表达序列标签(EST)进行了分析,筛选出了许多与雄性生殖调控相关的基因序列,也丰富了中华绒螯蟹的基因组资料。在此基础上,通过cDNA末端快速扩增(RACE)和实时定量PCR等技术进一步对Dmcl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雄性生殖的分子机制提供了研究基础。主要研究结果如下：1为了研究中华绒螯蟹精巢发育过程中储存的遗传信息,以精巢为材料构建了cDNA文库,文库的重组率为81.3%,库容量为3.5×106,插入片段大小为0.5-2kb。随机挑取了3,338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2,990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2,415条unique序列,包括307条重叠群(Contig)和2,108条单一序列(Singleton)。通过比对发现有922条Unique序列被注释。据序列相似性分析,发现了Dmcl、Vasa和HSP70等30个与生殖、发育相关的基因。通过精巢cDNA文库的构建及ESTs分析,初步明晰了与河蟹精巢发育相关的基因,为进一步研究中华绒螫蟹乃至十足类的功能基因组提供了重要的基础资料。2根据获得的Dmcl基因的EST序列,利用RACE技术克隆得到了中华绒螯蟹Dmcl基因cDNA全长序列。生物信息学分析发现,该序列全长为1,360bp,包括96bp的5’-末端非转录区(5’-UTR),1,026bp的开放阅读框(ORF)和238bp的3’-末端非转录区(3’-UTR),共编码341个氨基酸。分析表明该cDNA推导的氨基酸序列与甲壳类动物的同源性最高,和其它动物Dmc1蛋白序列也具有较高的同源性；Dmc1含有两个嘌呤核苷酸的结合位点(motifs A and B)和DNA结合区(L1and L2),与已报道其它物种的研究的结果一致；系统进化分析进一步的证实了该基因的保守性,与其它甲壳纲动物的亲缘关系最近,依次与其它相近物种进行聚类,符合了生物进化的特征。利用RT-PCR技术对中华绒螯蟹Dmcl组织特异性及其表达特征进行分析,发现在Dmcl基因在精巢中表达量最高,在卵巢、副性腺和血细胞中有微量表达,在心脏、肝胰腺、鳃、胃、肌肉、肠、胸神经节这几种组织中几乎不表达。采用实时荧光定量PCR技术检测河蟹Dmcl基因在精巢发育前期、中期、后期及末期四个不同发育阶段的表达丰度,结果表明Dmcl基因在前期的表达丰度最高,其次是中期和后期,在末期表达量最低。3采用抑制性差减杂交技术(SSH),分别以中华绒螯蟹发育前期精巢为检测方(tester),发育后期精巢为驱动方(driver)构建正向差减文库；以发育后期精巢为检测方(tester),发育前期精巢为驱动方(driver)构建反向差减文库。两个文库分别包括850个和700个cDNA克隆,重组率在90%以上；插入片段在250-1500bp之间。该河蟹精巢差减cDNA文库的建立为进一步在分子水平上阐明河蟹的雄性生殖调控机制奠定了基础。更多还原

【Abstract】 The Chinese mitten crab Eriocheir sinensis is one of the most important aquaculture species in China, its culture under facility conditions having started in the early1980’s. The annual output has increased during the past decade in China, from200,000tons in the year2000to420,000tons in2004. With the development of intensive culture, various problems have appeared in cultured populations, amongst others, sexual precocity. More and more individual crabs mature when small-sized. After sexual maturation, energy and nutrients are mainly diverted into gonad maturation or reproduction, with little left over for somatic growth, with the consequential devaluation of the commercial product and economic losses. Previous studies have revealed two main external factors as leading to sexual precocity in crabs, the environment and food. Nevertheless, few scientific investigations have been dedicated to elucidating the internal factors inducing sexual precocity. Furthermore, the regulative mechanisms of gonad maturation in E. sinensis at the molecular level are unknown.In the present study, a testis cDNA library of Chinese mitten crab Eriocheir sinensis-was constructed and ESTs were analyzed.30genes related to reproduction and development were discovered. cDNAs of Dmcl was gained basis on expressed sequence tags, followed by analysis of characterization and spatio-temporal expression. The results were as follows:1We constructed a cDNA library from the rapid developmental stage of testis of E. sinensis and sequenced3,388randomly picked clones. After processing,2,990high-quality expressed sequence tags (ESTs) were clustered into2,415unigenes including307contigs and2,108singlets, which were then compared to the NCBI non-redundant (nr) protein and nucleotide (nt) database for annotation with Blastx and Blastn, respectively. After further analysis,922unigenes were obtained with concrete annotations and30unigenes were found to have functions possibly related to the process of reproduction in male crabs-six transcripts relevant to spermatogenesis (especially Cyclin K and RecA homolog DMC1), two transcripts involved in nuclear protein transformation, two heat-shock protein genes, eleven transcription factor genes (a series of zinc-finger proteins), and nine cytoskeleton protein-related genes. Our results, besides providing valuable information related to crustacean reproduction, can also serve as a base for future studies of reproductive and developmental biology.2Dmcl is a protein involved specifically in the meiotic recombination during spermiogenesis of vertebrates, however, it is little characterized in lower invertebrates. In this study, a full-length cDNA coding Dmcl (termed EsDmcl) was cloned from testis of Chinese mitten crab Eriocheir sinensis by reverse transcript-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods. The cDNA obtained was1,360bp long with a1,029bp open reading frame (ORF) encoding341amino acids. The putative EsDmcl protein owns the Walker A (GPESSGKT), B boxes (VTVVD) and two disordered loops (denoted L1and L2motifs). Pairwise alignments demonstrated that EsDmcl had86%,78%,75%,75%and73%identity with its homologues in black tiger shrimp, black-legged tick, mouse, human and zebrafish, respectively. The phylogenetic tree revealed that EsDmcl is more related to invertebrate Dmcl than vertebrate. The EsDmcl transcripts were predominantly expressed in testis. Quantitative RT-PCR was used to determined the expression difference of EsDmcl gene in testis at earlier, middle, later and end stages of E. Sinensis. EsDmcl gene expression was highest at earlier development stage, and the next were middle and later stages. At end development stage, the expression level of EsDmcl gene was lowest. Results suggesting that differential expression of EsDmcl is closely related to spermatocyte meiotic maturation. Therefore, EsDmcl probably performs critical functions in the spermiogenesis of E. Sinensis.3In the present study, we performed suppression subtractive hybridization (SSH) experiments in the crab, Eriocheir sinensis, and constructed the forward subtractive cDNA library with cDNAs from the earlier stage (tester) and the later stage (driver) of the E. sinensis testis. The reverse subtractive cDNA library was constructed with cDNAs from testis during the later stage (tester) and the earlier stage (driver). The subtractive cDNA libraries were obtained including850and700clones respectively. By PCR detection, the length of the inserted fragments was250-1500bp on average. Further results indicated that the cDNA library quality are exactly within SSH library standard and could be used in further studies, also would provide reference for studying the differentially expressed genes to explore the molecular mechanism of testis, and regulation and control of spermatogenesis in E. sinensis.更多还原