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题名: 石斑鱼垂体EST分析及GTH的克隆和表达特征研究
作者: 李创举
答辩日期: 2007-06-16
导师: 桂建芳
授予单位: 中国科学院水生生物研究所
授予地点: 水生生物研究所
学位: 博士
关键词: 石斑鱼 ; 垂体 ; 性反转 ; 激素 ; GTHα ; FSHβ ; LHβ ; FSHR ; LHR
其他题名: EST analysis of the pituitary of the grouper and Cloning, identification and Characterization of GTH Genes in Groupers
摘要: 石斑鱼是重要的海产经济鱼类,在其个体发育过程中存在先雌后雄的天然性反转现象。人工养殖的环境下石斑鱼很少发生自发的性反转,因此,由于野生雄鱼不易获得,大规模人工养殖难以推广。垂体是调节生长和生殖等生理过程的重要内分泌器官。在本研究中,我们构建了分别处于卵巢发育起始和性反转后期斜带石斑鱼垂体SMART cDNA的质粒文库,并通过测序分别筛选到232个和258个EST。将所得EST与GenBank数据库中的序列进行比对,处于卵巢发育起始和性反转后期的斜带石斑鱼垂体EST中,激素所占比例均为最高,分别为40.5%和34.9%。进一步比较分析了这两个性腺发育时期斜带石斑鱼垂体EST中各种激素相对表达丰度。与处于卵巢发育起始时期相比,处于性反转后期的斜带石斑鱼垂体中的生长/催乳激素家族(GH、PRL和SL)、促甲状腺激素β亚基(TSHβ)和阿黑皮素原(POMC)表达水平下降;促性腺激素α亚基(GTHα)表达水平急剧上升,促滤泡激素β亚基(FSHβ)、促黄体激素β亚基(LHβ)表达水平上升。 从垂体SMART cDNA质粒文库中筛选到的EcGTHα、EcFSHβ和EcLHβ基因的全长cDNA分别为647、574和594bp,分别编码94、99和115个氨基酸残基的成熟多肽。EcGTHα、EcFSHβ和EcLHβ与其它鱼类GTH亚基有很高的同源性。RT-PCR和Western blot分析表明EcGTHα、EcFSHβ和EcLHβ仅在垂体中表达;EcGTHα和EcFSHβ在三个不同性腺发育时期斜带石斑鱼垂体中的表达基本相似,但EcLHβ显示出明显的表达差异。EcLHβ在卵巢发育起始期维持高水平表达,卵巢成熟期表达下降,最后在性反转后期表达量急剧上升。将这三种亚基分别进行体外表达并制备了多克隆抗体。Western blot结果显示,处于休眠期卵巢发育时期赤点石斑鱼的垂体蛋白中,EaGTHα亚基几乎检测不到,EaFSHβ和EaLHβ亚基的表达量极低;而处于发育中卵巢时期赤点石斑鱼的垂体中GTH三个亚基的表达均上升到较高的水平。赤点石斑鱼垂体切片免疫荧光定位研究和血清ELISA同样得出了与Western blot分析相似的结论。以上多种技术研究了GTH三个亚基在石斑鱼不同性腺发育阶段的时空表达图谱,揭示出石斑鱼GTHα、FSHβ和LHβ不同于其它鱼类的表达特征。免疫荧光定位研究显示GTHα定位于石斑鱼垂体PPD区域、PI区域外围和RPD区域;FSHβ定位于PPD区域;LHβ定位于PPD区域和PI区域外围。双色荧光标记和激光共聚焦显微镜观察,清晰地将石斑鱼FSHβ和LHβ定位于垂体同一区域的不同细胞群中,为解决FSHβ和LHβ是否由同一细胞分泌这个长期争论的问题提供了确凿的细胞学证据。 鱼类存在两种GTH受体(GTHR),FSHR和LHR。本研究采用RACE-PCR方法,从赤点石斑鱼性腺中克隆了EaFSHR的全长cDNA和EaLHR的部分cDNA。EaFSHR cDNA的多聚腺苷酸尾前的序列长2393bp,编码701个氨基酸。EaFSHR多肽由信号肽(21aa)、胞外区(355aa)、跨膜区(264aa)和胞内区(61aa)构成。EaLHR仅获得跨膜区部分的cDNA。EaFSHR与其它鱼类有很高的同源性。RT-PCR结果表明EaFSHR为性腺特异表达,而EaLHR除了性腺中的表达外,在肝和脾中也有表达。
英文摘要: EST analysis of the pituitary of the grouper and Cloning, identification and Characterization of GTH Genes in Groupers ChuangJu Li (Genetics) Directed by Prof. JianFang Gui Abstract The grouper is an important marine aquaculture fish and has been cultured in China and Southeast Asian countries. However, large-scale aquaculture of the grouper has been hindered by the rarity of natural males because it is a protogynous hermaphroditic fish and sex inversion rarely occurs in captive stocks. In teleost, pituitary plays significant roles in many physiological functions, such as growth and reproduction. In this study, the SMART cDNA plasmid libraries of the pituitaries from orange-spotted grouper at the early stage of ovary development and at the late stage of sex inversion were constructed respectively. 232 and 258 ESTs were obtained respectively from the two pituitary plasmid libraries. Among the two ESTs, the genes homologous to hormones are both the largest group, which accounts for 40.5% and 34.9% respectively. Compared with the express level of hormones in pituitary at the early stage of ovary, the express levels of GH, PRL, SL, TSHβ and POMC were declined, GTHα, FSHβ and LHβ were rised at the late stage of sex inversion. The nucleotide sequences of EcGTHα, EcFSHβ and EcLHβ subunit cDNAs are 647, 594, and 574 bp in length, and encode for mature peptides of 94, 99 and 115 aa respectively. High homology were observed by amino acid sequence alignment and identity comparison of the grouper mature peptides of EcGTHα, EcFSHβ and EcLHβ with that of other fishes. Phylogenetic tree analyses of the three GTH mature subunits revealed similar phylogeny relationships among the studied fish species. RT-PCR analysis and Western blot detection demonstrated pituitary-specific expression of EcGTHα, EcFSHβ and EcLHβ. Furthermore RT-PCR analysis showed that the pituitary EcGTHα and EcFSHβ mRNA expression levels were almost similar at the three different stages of gonad development, however, the EcLHβ mRNA level showed obvious expression difference, suggesting that EcLHβ might keep higher level at the stage of oogenesis, then decrease in ovary maturation stage, and finally abruptly increase during sex inversion stage. Three polyclonal antibodies were prepared from the in vitro expressed EcGTHα, EcFSHβ and EcLHβ mature peptidess respectively. Western blot analysis, ELISA and immunofluoresence localization were performed on two typical stages of ovarian development stages in red-spotted grouper. Significant differences in protein expression levels of three GTH subunits were revealed between the two ovarian development stages. In the individuals with resting ovary, EaGTHα was almost not detected in pituitaries, and EaFSHβ and EaLHβ expression levels were very low. While in the individuals with developing ovary, the expression of all three GTH subunits reached to a high level. Similar results were gained by ELISA and immunofluoresence localization. Immunofluoresence localization indicated that the grouper FSHβ cells mainly distributed in the middle area of PPD, while the LHβ cells distributed more widely, including in the area similar to the FSHβ cells and at the external periphery of pituitary near to the PI side. The GTHα might be expressed in both FSHβ and LHβ cells. Double immunofluoresence localization further demonstrated FSHβ and LHβ expression in distinct cells in the PPD area, although the FSHβ and LHβ cells were detected in the identical area of PPD. The full length cDNA of FSH receptor (FSHR) and partial cDNA of LH receptor (LHR) were cloned and sequenced from the gonad SMART cDNA library of red-spotted grouper. The nucleotide sequences of EaFSHR cDNA is 2393 bp in length, and encodes for peptides of 701 aa. High homology was observed by amino acid sequence alignment and identity comparison of the grouper peptide of EaFSHR with that of other fishes. Phylogenetic tree analyses of the EaFSHR revealed similar phylogeny relationships among the studied fish species. RT-PCR analysis revealed goand-specific expression of EaFSHR. In addition to gonad, the expression of EaLHR could be detected in the liver and spleen.
语种: 中文
内容类型: 学位论文
Appears in Collections:中科院水生所知识产出(2009年前)_学位论文

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