【作者】 曹谨玲；

【导师】 夏德全； 吴婷婷；

【作者基本信息】 南京农业大学 ， 水生生物学， 2007， 博士

【摘要】 罗非鱼是原产于非洲的热带鱼类，现已成为世界性的主要养殖鱼类。在生产上，罗非鱼雄鱼比雌鱼生长快40-50％，因此提高罗非鱼雄性率具有重要实践意义。在各种罗非鱼杂交组合中，奥尼杂交鱼的雄性率最高，为95％，但根据染色体决定性别理论，其雄性率应为100％。由于鱼类的性别决定机制是多样的和易变的，较高等动物要复杂得多，因而通过分子生物学的研究从基因水平进行探讨，是一条揭示罗非鱼性别控制机理的好途径，为进一步提高罗非鱼雄性率奠定基础。本研究利用RT-PCR和RACE方法克隆了奥利亚罗非鱼的DMO和DMT基因，并对它们的表达特性进行了研究，这将有助于了解奥利亚罗非鱼的性别决定机制，从而采取人为控制措施，对奥利亚罗非鱼和尼罗罗非鱼杂交产生全雄杂交后代的理论研究和生产实践具有重要意义。利用RT-PCR和cDNA末端快速扩增法分别从奥利亚罗非鱼(Oreochromis aurea)卵巢和精巢分离、克隆DMO和DMT基因，并进行序列测定与生物信息学分析。结果表明，DMO基因cDNA序列全长1571 bp [不含poly(A)]，包括148 bp 5’非翻译区，1230 bp阅读框以及含Poly(A)信号AATAAA的193 bp3’非翻译区[不包括Poly(A)]，阅读框共编码409个氨基酸，与尼罗罗非鱼DMO进行比较，同源性为96．3％，说明DMO在同物种间差别较小。而与尼罗罗非鱼，红鳍东方豚，虹鳟，青鳉，鼠，人等动物的DMRT1进行比较，同源性分别为：25．7％，25．8％，24．3％，29．7％，22．5％，22．0％。DMT基因cDNA序列全长1260 bp，包括74 bp 5’非翻译区，879 bp阅读框以及含poly(A)信号AATAAA的307 bp 3，非翻译区，阅读框共编码292个氨基酸。序列同源性分析表明，不同进化地位动物的DMRT1基因DM域编码序列存在高度同源性，显示DMRT1基因在系统进化上高度保守。生物信息学分析结果表明，DMO蛋白具有两个亲水性螺旋卷曲区域，97～112，155～168氨基酸区域，没有信号肽，含有两个跨膜结构域，发生跨膜运动。DMT蛋白不含螺旋卷曲区域，没有信号肽，是一个非跨膜的亲水性稳定蛋白，该蛋白以游离形式存在于细胞质内，不会发生跨膜运动。DMO和DMT包含两个相同的保守的功能结构域，分别行使性别调控，使DNA形成二聚体和结合回纹结构的功能。DMO和DMT都含有多个磷酸化位点，推测它们可能在细胞信号传导中发挥作用，且其生物活性可能接受信号途径中多种信号的调控。DMO蛋白N-端第1～5，41～51，65～67，86～89，98～110，154～170，183～203，205～248，258～264，284～291，293～298，270～375，389～392，402～410区域和DMT蛋白N-端第1～9，17～28，77～84，114～123，131～139，157～184，196～207区域可能是B细胞表位优势区域。DMO和DMT的高级结构相似，都含有两个a-螺旋区域。采用荧光定量RT-PCR方法，从mRNA水平对奥利亚罗非鱼DMO和DMT基因的时空表达谱进行了研究。结果发现，这两个基因均从原肠胚早期开始转录，一直到出膜，都维持着较高的表达水平，但DMO表达量明显高于DMT。鱼苗性腺分化时期，激素处理可显著提高奥利亚罗非鱼的雌、雄比率，还可影响DMO和DMT基因的表达量，提示它们可能与激素调控有关。在雌、雄鱼的肝和肾等5种组织中均检测不到这两个基因转录本的存在；在脑中仅可检测到DMO基因不同强弱的转录本，呈现出很强的中枢神经系统的表达特异性。此外，在成体的卵巢和精巢中分别只可检测到DMO和DMT基因的大量表达，显示二者在性别决定和分化中的重要功能。奥利亚罗非鱼DMO基因在其中枢神经系统发育及卵巢发生和功能维持上有着重要功能；DMT基因在精巢发生和功能维持上起重要作用。为了进一步分析这两种基因mRNA在胞内的表达情况，利用原位杂交分析了奥利亚罗非鱼端脑、下丘脑、垂体、性腺、肝脏、心脏、脾脏、肾脏、肌肉等组织中DMO和DMTmRNA的表达情况，结果表明：DMO只在奥利亚罗非鱼的卵巢中表达；DMT仅在其精巢中表达。在奥利亚罗非鱼卵子发生中，DMO mRNA均匀分布于卵原细胞和各期卵母细胞的胞质中；在卵原细胞和Ⅰ、Ⅱ期卵母细胞中，随着卵母细胞的发育，DMO mRNA的杂交信号逐渐增强，最后充满整个胞质中。Ⅲ期卵母细胞中，DMO均匀分布在卵黄颗粒之间的细胞质中．Ⅳ期以上的卵母细胞胞质中，相对于前三个时期DMO mRNA的杂交信号明显减弱，整个胞质都呈现较淡的黄色，有向胞质外周皮质层迁移集中的趋势。当卵黄充满整个胞质之后，在整个卵母细胞中无DMO mRNA的杂交信号。在奥利亚罗非鱼精子发生中，DMT mRNA可在精原细胞和初级精母细胞中检测到。DMT mRNA的阳性信号在精原细胞中极为强烈，在初级精母细胞中较为微弱，而精子细胞中没有阳性信号。结果初步表明，奥利亚罗非鱼DMO和DMT基因对于生殖干细胞。卵原细胞和精原细胞的维持和正常功能可能起着重要作用。为了进一步探讨奥利亚罗非鱼DMO和DMT的功能，本研究采用RT-PCR方法分别从奥利亚罗非鱼卵巢和精巢中克隆出DMO和DMT全长cDNA片断，构建了pMAL-c2x／DMO和pMAL-c2x／DMT重组质粒，成功地表达了DMO和DMT蛋白，发现在IPTG诱导4h后，目的蛋白可达到细菌总蛋白的50％左右。同时通过免疫印迹技术证明了这两种蛋白的免疫原性。经Xa切割、Amylose-sepharose柱层析纯化后作为抗原免疫新西兰白兔制备了DMO和DMT多克隆抗体，并进行纯化。通过对纯化多抗进行Westem blot分析，结果表明获得了高特异性的DMO和DMT抗体。为了观察DMO和DMT在组织中的表达谱，首先，本研究制备了多种组织匀浆蛋白，使用纯化的抗体进行Western blot分析，仅分别在卵巢和精巢中检测到DMO和DMT蛋白的表达；制备奥利亚罗非鱼多种组织切片，使用纯化的DMO和DMT多抗进行免疫组织化学分析，发现DMO仅在卵巢表达，而DMT仅在精巢表达，但其特异性低于原位杂交。以上结果有助于阐明DMO和DMT的功能及在鱼类性别调控中的作用．本研究首次从奥利亚罗非鱼中克隆到性别调控基因DMO和DMT，并分析了它们的表达特征及可能的功能。奥利亚罗非鱼DMO和DMT基因的成功克隆及分子生物学特征和功能分析不仅为DMRT基因的分子进化和相似性比较研究提供了新的材料，而且对于进一步研究鱼类性别调控及DMRT基因的结构和功能有着重要的理论价值和研究前景。更多还原

【Abstract】 Tilapia is native to Africa but introduced elsewhere as a valuable food fish. It is very popular all over the world. In aquaculture, the male grows more quickly than the female by 40-50%, so it is very important to improve the male rate. In the hybrid combinations of various cichlid fishes of the genus Tilapia, the male rate of offspring of Oreochromis aurea(♂)×Oreochromis niloticus(♀) is the highest(95%), but it should be 100% according to the chromosomal theory. Sex determination and differentiation in fish is highly variable and complicated, so molecular biology methods are used necessarily to reveal the sex regulatory mechanism and improve the male rate in tilapia at gene level.The DMRT genes constitue a new gene family related to sex-determination. Like the Double-sex gene of Drosophila melanogaster and the Mab-3 gene of Caenor habditis elegents, they encode transcription factors characterized by a conserved zinc-finger like DNA-binding motif, the DM domain, which is thought to bind DNA in the process of sex differentiation and development. In 1998, DMRT1 genes were found to regulate sex determination and differentiation in vertebrates extensively. So far, the DMRT genes have been discovered in a wide range of animal species, such as fish, amphibian, reptiles, birds and mammals. These evidently reveal the evolutionary conservation of DMRT gene family.The DM domain gene family has multiple members in both invertebrates and vertebrates. For example, up to date, there are at least seven DM domain gene found in mouse, eight in human, four in Drosophila, eleven in C. elegans, and six in fish (DMRT1-5 and DMRT2b). All of them encode putative transcription factors related to the sexual regulator Dsx of Drosophila and Mab-3 of C. elegans. Multiple DM domain genes have been suggested to be involved in mouse sexual development. At least three mouse DM domain genes in addition to DMRT1 are expressed in embryonic gonad, including DMRT3, DMRT4, and DMRT7. However, DMRT2 is expressed in presomitic desoderm and development somites, while DMRT5 and DMRT6 are expressed primarily in the brain, suggesting a role in other developmental precesses.Although some of the DM genes are involved in sexual development, function of most of these genes remains unclear, and we know remarkable little about the evolution of the DM genes. It is still the question that the male-specific role of the DM gene is primordial during their evolution, or independently evolved by convergence. Furthermore, it is essential to understand the roles of the DM genes in regulatory pathway of sex determination, or if any, the roles in other developmental processes.There is also few structural and functional analysis concerning DM domain genes of the model fish Oreochromis aurea. We report here cloning, characterization and expression of DMO and DMT of Oreochromis aurea.In this study, RT-PCR and RACE were used for the cloning of DMO (DM-domain gene in ovary) and DMT (DM-domain gene in testis) full length cDNA from ovary and testis of Oreochromis aurea, respectively. DMO and DMT genes were sequenced and analyzed by bioinformatics methods. Sequence analysis revealed a 1571 bp cDNA full-length sequence of DMO containing 148 bp 5’-untranslated region, 193 bp 3-untranslated region and 1230 bp ORF encoding 409 amino acid. Homology of DMO from Oreochromis aurea and Oreochromis niloticus was 96.3%. However, we compared the alignment of deduced amino acid sequences between DMO cDNA from Oreochromis aurea and DMRT1 cDNA from Oreochromis niloticus, fugu, rainbow trout, medaka, rat to human. The score was 25.7%, 25.8%, 24.3%, 29.7%, 22.5% and 22.0%, respectively. A 1260 bp cDNA full-length sequence of DMT encoded 292 amino acids, which contained 74 bp 5’-untranslated region, 307 bp 3’-untranslated region and 879 bp ORF. The deduced amino acid sequence aligned with those of DMRT1 genes from different species, high sequence homologies were obtained as revealed in phylogenic tree constructed.The amino acid sites of DM domains may form C2/H2 model zinc-finger structure to bind specific DNA sequence and regulate sex differentiation and development. DMT contained a male specific motif, which was well conserved among numerous DMRT1 genes, but was absent in DMO indicating that DMT represented a male-type DM-domain gene and played an important role in sex differentiation and development.The bioinformatics analysis revealed that DMO had two helical segments that were 97-112 amino acid sequence and 155-168 amino acid sequence, and did not contained signal peptide. It was a transmembrane and hydrophilic protein. DMT had not helical segment and did not contained signal peptide. It was a non-transmembrane and hydrophilic protein. DMO and DMT included two same functional domains, which played the roles of sex control, dimerising and binding palindromic DNA, respectively. DMO and DMT both included several phosphorylation sites implicating that they could play some roles during cellular signal conduct and their activities might be related to the regulation of many signals during signal route. DMO and DMT had similar advanced structures including two a-helix regions. Moreover the B-cell epitopes possibly localized in or nearby the DMO protein’s N-termianl No.1-5, 41-51, 65-67, 86-89, 98-110,154-170,183-203, 205-248, 258-264, 284-291, 293-298, 270-375, 389-392 and No.402-410, and DMT protein’s N-termianl No.1-9,17-28, 77-84,114-123,131-139,157-184 and No.96～207.The temporal and spatial expression patterns were analyzed by Real-time Quantitative RT-PCR at cellular level. Their transcripts appeared from early gastrulae stage during embryonic development, and maintained a considerable high level till the one day’s fry, but level of DMO was higher than that of DMT. Treatment with hormone not noly improved remarkably the female or male rate, but also changed level of DMO and DMT during sex differentiation, implicating that they could be related to hormone regulation. No DMO and DMT transcripts were found in liver, kidney, spleen, heart and muscle, but unequal amount of DMO transcripts were detected in both brains tissues of female and male Oreochromis aurea, which suggested that mRNA expression of DMO was specific in central nervous system (CNS). In addition, we also found the abundant transcripts of DMO in ovary and DMT in testis. This supports that the DMO and DMT genes play important roles not only on the sex determination, but also on the development processes of early embryogenesis. Based on these results, we suggest that DMO should play a key role in CNS and ovary development of Oreochromis aurea and DMT in testis. Study on DMO and DMT expression facilitates the elucidation of the roles of them and the understanding of sex differentiation and development in fish.Their mRNA expression was further analyzed at cellular levels using in situ hybridization. The results were as follows: DMO was expressed only in ovary and DMT was observed in testis exclusively. DMO mRNA uniformly dispersed throughout the cytoplasm of oocytes at all stages. With the development of oocytes in oogonia and stage I、II oocytes, the expression signal became stronger. In stage of III oocyte, DMO mRNA was uniformly observed in cytoplasm between grains of yolk. In the later stage, the signals of DMO mRNA significantly decreased in most of the regions while remained strong at cortical region. DMO mRNA signals were not detected throughout the oocytes after the yolk was full of the cytoplasts. During spermatogenesis, the positive signals of DMT mRNA could only be detected in spermatogenia and primary spermatocytes while the signals in the former were much stronger than in the latter. Howerer, no signals could be detected in spermatids. The results suggest that DMO and DMT may play an important role in maintenance and functioning of the gernline stem cell-oogonia and spermatogonia.To further inquire into the function of DMO and DMT, the intact regions encoding DMO and DMT obtained by RT-PCR were sub-cloned into the vector pMAL-c2x prokaryotic expression system and introduced into the Escherichia coli TB1 cell for efficient fusion expression. It was found that the expression level was about 50% of total protein in the engineered bacteria after IPTG induction for 4h. Immunoblotting proved the immunogenicity of DMO and DMT. After purification and cleavage, DMO and DMT proteins were used to immunize the adult rabbits following standard protocols. Consequently, we found that polyclonal antibodies against DMO and DMT had high specificity by Western blot analysis.The expression of DMO and DMT protein was also analyzed using the purified antibodies through Western blot and immunohistochemistry. We found DMO was exclusively expressed in ovary and DMT in testis, but specificy by immunohistochemistry is lower than by in situ hybridization. Study on DMO and DMT expression facilitates the elucidation of the roles of them and the understanding of sex differentiation of fish.In general, this is the first time to clone DMO and DMT genes related to sex control from Oreochromis aurea. The cloning, molecular characterization and function of DMO and DMT in Oreochromis aurea provide not only new materials for researches on DMRT molecular evolution and similarity comparison, but also theoretical basis for better understanding the sex control and DMRT in fish.更多还原