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银鲫卵细胞特异表达的组蛋白H2A变体及nucleophosmin的研究
Alternative TitleStudy of Oocyte-specific Histone H2A Variant and Nucleophosmin in Gibel Carp
吴南
Subtype博士
Thesis Advisor桂建芳
2008-06-17
Degree Grantor中国科学院水生生物研究所
Place of Conferral水生生物研究所
KeywordCagoh2a基因 Cag-nucleophosmin基因 Ca-nucleophosmin基因 表达模式 蛋白动力学 银鲫 彩鲫
Abstract银鲫有着天然的雌核发育现象,这与其卵子的成分的特殊性是分不开的。本论文在实验室前期工作的基础上,着手研究银鲫成熟卵母细胞中的大量表达的两个基因——组蛋白H2A变体和nucleophosmin。本研究通过描述银鲫的卵细胞特异的组蛋白H2A变体,以及银鲫和其对照物种彩鲫的性腺特异的nucleophosmin基因的分子特征和表达模式,以及蛋白动力学等基本特征,试图从分子机理上揭示银鲫的配子发生和早期胚胎发育的特殊性。 银鲫的卵巢中有一种特殊的组蛋白H2A的变体——CagOH2A。本研究的第一方面,用序列比对和系统聚类树分析了CagOH2A和一个体细胞组蛋白 CagH2A的分子特征。第二方面从RNA和蛋白水平研究了CagOH2A在各种组织和胚胎发育过程中的时空表达模式,发现CagOH2A蛋白是仅在成体的卵巢组织中和原肠期以前的早期胚胎中存在;并用原位杂交的方法在卵巢中检测到CagOH2A的mRNA只在各种卵细胞中存在,而在周围的体细胞中不存在;所有Western blot检测到的CagOH2A蛋白的大小都暗示着其可能发生单泛素化,而更为细致的Western blot分析CagOH2A蛋白在卵子发生和成熟过程中的蛋白带型,发现在此过程中CagOH2A蛋白有可能发生某种修饰,根据哺乳类卵子发生过程中常见的组蛋白修饰,推测为甲基化。第三方面采用FRAP实验方法研究了CagOH2A蛋白在核小体中的动力学特征,并用了一种体细胞的组蛋白H2A——CagH2A作为分析的对照,揭示了CagOH2A蛋白的N/C端区域的特殊的结合性质。我们的研究结果表明CagOH2A是一个卵细胞和早期胚胎特异的组蛋白变体,其更为游离的N端可能提供了更多的修饰位点,并能轻微降低核小体的稳定性,而其紧密结合的C端保证了核小体的中心区域的稳定。 我们在银鲫和彩鲫中都克隆得到nucleophosmin/nucleoplasmin家族的一个成员,分别命名为Cag-nucleophosmin和Ca-nucleophosmin,比对了序列的同源性,并预测了磷酸化位点。还采用Western blot和免疫荧光定位的方法分析了Cag-nucleophosmin和Ca-nucleophosmin蛋白各自的表达特征,如下:在成体各组织中都是性腺特异表达,只在精巢和卵巢中检测得到,在精巢中仅在精原细胞中有强烈信号,在初级精母细胞中有微弱的信号,而在后期分化的精细胞中Cag-nucleophosmin和Ca-nucleophosmin都没有表达;在卵巢中,Cag-nucleophosmin和Ca-nucleophosmin在各种卵细胞中都有表达,但在周围的体细胞中无表达,且进一步的Western blot分析Cag-nucleophosmin和Ca-nucleophosmin在卵子发生和成熟过程中的蛋白带型,发现在此过程中二者都可能发生某种修饰,根据其他nucleophosmin/nucleoplasmin蛋白在卵子发生过程中的修饰,推测为磷酸化;在胚胎中Cag-nucleophosmin蛋白和Ca-nucleophosmin蛋白都一直存在,且蛋白带都为不变的单带。综上,它们在银鲫和彩鲫中的分子和表达特征相似,未发现显著差别。
Other AbstractNatural gynogenesis,the special reproduction mode of gybel carp(Carassius auratus gibelio),is closely related to the specific components in its oocytes. In this thesis, we focus on two genes expresed in abundant in fully-grown oocytes of gybel carp,one is an ovary-specific histone H2A variant,the other is nucleophosmin.In order to reveal the molecular mechanism of gametogenesis and early embryonic development of gybel carp, we demenstrate the charateristics of the two genes in many aspects,like molecular charaterization, expression pattern and protein dynamics. CagOH2A is the ovary specific histone H2A variant in gybel carp. In this study, firstly we analyzed molecular charateristics of CagOH2A and a somatic histone H2A CagH2A using sequence alignment and phylogenetic tree.Secondly, we investigated the temporal and spacial expression pattern of CagOH2A in various tissues and embryonic development from both RNA and protein aspects. We found CagOH2A existed only in ovary and early embryos before gastrula, and also found that its mRNA only located in oocytes of every stages in ovary using in situ hybridization. However,All the bands shown in Western blot are not the size of histone H2A, but the size of monoubiquitinated histone H2A, so we suspect monoubiquitination of CagOH2A may occur.Moreover, the result of more precise Western blot analysis of CagOH2A expression during oogenesis and oocyte maturation indicates another kind of modification occures in this process. We guess it may be methylation based on histone modifications during oogenesis in mammals.Thirdly, we did FRAP experiments to reveal the dynamics of CagOH2A in nucleosome with a somatic histone H2A, CagH2A, as control.The results reveal the relatively lower stability of nucleosome containing CagOH2A, and the specific binding dynamics of N/C terminal of CagOH2A,as that its N terminal is more relax and may contain more sites for modifications,and its C terminal binds to nucleosome more tightly than somatic histone H2A. And in this thesis,one member of nucleophosmin/nucleoplasmin family was also isolated and charaterized in both gybel carp and color carp,called Cag-nucleophosmin and Ca-nucleophosmin respectively. We analyzed their molecular charateristics using sequence alignment and phosphorylation sites prediction,and also analysed expression pattern using Western blot and immunohistochemistry. The result shows that both of them express in gonad specificly among various tissues.In testis, the signal is strong in spermatogonia, weak in primary spermatocytes, and not found in other sperm cells or somatic cells.And in ovary, the signal exists all through oogenesis, in oocytes of all stages, but not exists in somatic cells. Further Western blot analysis during oogenesis and oocyte maturation indicates certain modification of both Cag-nucleophosmin and Ca-nucleophosmin occurs in oocyte maturation. We suspect it may be phosphorylation based on the research of other nucleophosmin/nucleoplasmin. In embryogenesis, both Cag-nucleophosmin and Ca-nucleophosmin exist in every stage with one singal unchanged band. Therefore, the above observations show no significant difference between Cag-nucleophosmin and Ca-nucleophosmin in molecular and expressional aspects.
Pages113
Language中文
Document Type学位论文
Identifierhttp://ir.ihb.ac.cn/handle/342005/12286
Collection学位论文
Recommended Citation
GB/T 7714
吴南. 银鲫卵细胞特异表达的组蛋白H2A变体及nucleophosmin的研究[D]. 水生生物研究所. 中国科学院水生生物研究所,2008.
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