|Other Abstract||Natural 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.|