【作者】 马长艳；

【导师】 周开亚；

【作者基本信息】 南京师范大学 ， 动物学， 2003， 博士

【摘要】 第一部分 中华绒螯蟹卵巢发育相关基因的筛选 1 中华绒螯蟹卵巢差减cDNA文库的构建 应用抑制性差减杂交技术，构建了中华绒螯蟹卵巢两个发育时期的差减cDNA文库。其中以Ⅲ期卵巢为试验方（tester），Ⅱ期卵巢为驱动方（driver）进行正向差减杂交；以Ⅱ期卵巢为试验方，Ⅲ期卵巢为驱动方进行反向差减杂交。分别将所获差减cDNA片段插入质粒表达载体（PinPoint Xa-1 T-Vector），转化大肠杆菌JM109。最后所获正向差减文库含863个重组子，反向差减文库含360个重组子。PCR扩增鉴定正、反向差减cDNA文库的插入片段平均大小分别为360bp和160bp，表明所构建的差减文库适合进一步研究中华绒螯蟹卵巢发育相关基因。 2 用cDNA微阵列筛选中华绒螯蟹卵巢发育相关基因 将差减cDNA文库中的所有克隆用碱裂解法提取质粒，然后以提取的质粒为模板，用PCR方法扩增插入片段。PCR产物经浓缩、变性处理后，用自动点样仪点制于尼龙膜载体上，制成cDNA微阵列。分别将中华绒螯蟹Ⅱ期和Ⅲ期卵巢的mRNA反转录标记成³³P探针，然后将2种探针分别与2张相同的膜杂交，经洗膜、压片和放射自显影后进行信号的扫描和分析。结果共获得2倍以上差异表达克隆167个。其中Ⅲ期高表达克隆104个，Ⅱ期高表达克隆63个。 3 中华绒螯蟹卵巢发育相关基因cDNA片段的序列测定 从cDNA微阵列筛选的差异表达克隆中选取部分克隆进行测序。结果正向差减文库中获得7个独立的EST。同源性分析结果表明，这些EST皆为新的EST，已被dbEST收录，检索号分别为：CA591892、CA591893、CA591894、CA591895、CA591896、CA591897和CA591898。 南京师范大学博士研究生毕业论文第二部分 中华绒螫蟹卵巢发育相关基因全长叨＊A的克隆及序列分析工 中华绒蟹蟹卵巢RA＊kC＊＊A文库的构建 为了获得差异表达基因的全长CDNA序列，运用SMART技术，构建了中华绒蟹蟹卵巢（ill期）RACE CDNA文库。琼脂糖凝胶电泳结果表明，文库所含全长 CDNA的长度主要集中在 500～2 000 hp之间，RACE PCR结果表明，所用基因特异性引物与接头引物皆能扩增出产物，说明构建的文库质量较好，适于用RACE方法从中分离中华绒螫蟹卵巢发育相关基因的全长。DNA。二中华绒螫蟹卵巢发育相关基因E＊0且的全长CO＊A克隆和序列分析 根据SSH结合CDNA微阵列技术获得的EST004＊bEST检索号：CA591895)的序列设计引物，分别用 5’RACE和 3RACE的方法获得了 EJO基因的全长cDNA序列。同时用生物信息学的方法对 EJO基因的结构和功能进行了初步分析；用RTPCR的方法对该基因的表达谱进行了研究；用NOrtheffi印迹分析的方法对该基因在＊期和＊期卵巢的表达差异性进行了进一步验证。结果表明获得的EJOI基因的CDNA长度为876 hp，开放阅读框长度为759 hp，编码252个氨基酸。由氨基酸序列推断的 EJO基因编码蛋白的等电点为 6．18，分子量为28．18 kDa石wissProt数据库中未发现与 EJOI基因编码蛋白有意义的匹配序列。新基因 EJO编码的蛋白可能为分泌蛋白，前 17个氨基酸为信号肽。EJO基因在中华绒赘蟹的心脏、肠和卵巢中皆有表达，而在肌肉和肝胰腺中未见表达。Northern印迹分析结果表明EJOI基因在m期卵巢高表达。EJOI基因的GenBank检索号为：AY185917。3中华城螫蟹卵巢发育相关基因EJ 03的全长0＊A克隆和序列分析 根据SSH结合CDNA微阵列技术获得的EST003（dbEST检索号：CA591894）的序列设计引物，用5’RACE和3’RACE的方法获得了EJO3基因的全长cDNA序列。同时用生物信息学的方法对EJO3基因的结构和功能进行了初步分析；用RTPCR的方法对该基因的表达谱进行了研究；用N。them印迹分析的方法对该基因在＊期和皿期卵巢的表达差异性进行了进一步验证。结果表明获得的EJO3基因的 CDNA长度为 1514 hp，开放阅读框长度为 1368 hp，编码 456个氨基酸。由氨基酸序列推断的 EJO3基因编码蛋白的等电点为 5．79，分子量为 50刀0 ica。SwissProt数据库中未发现与EJO3基因编码蛋白有显著性意义的匹配序列。新基因EJO3编码的蛋白可能为分泌蛋白，前20个氨基酸为信号肽。EJO3基因主要存在2个转录本，其中较大的转录本除了在中华绒资蟹卵巢中表达外，在肠 3 南京师范大学博士研究生毕业论文中也有弱表达，而在心脏、肌肉和肝胰腺中未见表达；较小的转录本在所研究的组织中皆有表达。此外，在肌肉组织中除了存在较小的转录本外，还有2个较弱的转录本。Nonilern印迹分析结果表明EJO3基因在＊期卵巢高表达。EJO3基因的GenBank检索号为：AY185919。更多还原

【Abstract】 Part 1 Screening of genes related to the ovary development of the mitten crab (Eriocheir japonica sinensis)Chapter 1 Construction of subtractive cDNA library of mitten crab (Eriocheir japonica sinensis) ovaryTwo subtracted cDNA libraries of mitten crab (Eriocheir japonica sinensis) ovaries at two successive developmental stages were constructed by suppression subtractive hybridization (SSH). Two-directional (forward and backward) SSH was performed on cDNAs of the mitten crab ovaries which were either at stage II or at stage III. Two-directional subtracted cDNA fragments were cloned into PinPoint plasmid vectors, and the vectors were transformed into E. Coli JM109. Finally, the forward and backward subtracted cDNA libraries including 863 and 360 clones respectively were obtained. The length of the inserted fragments of the forward and backward subtracted cDNA libraries was 360 and 160 base pairs in average respectively by PCR detection. The results showed that the subtracted libraries constructed were suitable for further study on the genes related to the ovary development of the mitten crab.Chapter 2 Screening of genes related to the ovary development of the mitten crab (Eriocheir japonica sinensis) by cDNA macroarray analysisPlasmids were extracted from all the clones in the subtracted cDNA libraries with alkali lysis method, then the inserted fragments in plasmids were amplified by PCR. cDNA macroarray was made as follow: products of PCR were condensed and denatured prior to being robotically printed onto nylon membrane. mRNA from the mitten crab ovaries at stages II and III were made as 33P probes by reversetranscription reactions respectively, then two identical membranes were hybridized with the two kinds probes. Washing, pressing and autoradiography were performed before signals scanning and analysis. In total, 167 clones whose signal difference was >2 fold were obtained. Of the differentially expressed clones, 104 clones were highly expressed in the ovary at stage III, the remaining clones were highly expressed in the ovary at stage II.Chapter 3 Sequencing cDNA fragments of genes related to the ovary development of the mitten crab (Eriocheir japonica sinensis)Partial clones were sequenced which were selected from the differentially expressed clones screened by cDNA macroarray analysis. Seven independent ESTs were obtained from the forward subtracted cDNA library. Homology analysis showed that all the ESTs were novel ESTs, and all of them were accepted by dbEST. The accession numbers are: CA591892, CA591893, CA591894, CA591895, CA591896, CA591897 and CA591898.Part 2 Cloning and analysis of the full-length cDNAs of genes related to the ovary development of the mitten crab (Eriocheir japonica sinensis)Chapter 1 Construction of RACE cDNA library of mitten crab (Eriocheir japonica sinensis) ovaryIn order to get full-length cDNA sequences of the differentially expressed genes, a RACE cDNA library of the mitten crab ovary at stage III was constructed successfully with SMART technique. The result of the agarose gel electrophoresis showed that the length of the full-length cDNAs in the library was pooled mainly between 500 and 2 000 base pairs. By RACE PCR, amplified products were obtained with all the gene-specific primers and the adaptor primers. This verified that the quality of the RACE cDNA library was high. It was appropriate for cloning the full-length cDNAs of the genes related to the ovary development of the mitten crab.Chapter 2 Cloning and analysis of the full-length cDNA of EJO1 gene related to the ovary development of the mitten crab (Eriocheir japonica sinensis)Primers were designed according to the sequence of EST004 (dbEST accen-ssion number: CA591895) which was gotten using SSH and cDNA macroarrayapproach. The full-length cDNA sequence was finally generated by 5’RACE and 3’RACE respectively. At the same time, structure and function of EJOl gene were primarily analyzed by bioinformatics method. RT-PCR was made to study the expression profile of EJO更多还原