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Thesis Advisor桂建芳
Degree Grantor中国科学院水生生物研究所
Place of Conferral中国科学院水生生物研究所
Degree Discipline水生生物学
Keyword雌核发育 银鲫卵母细胞成熟 差减杂交
Abstract本研究采用差减杂交比较雌核发育银鲫(Carassius auratus gibelio)与两性生殖彩鲫(Carassius auratus color variety)完全长成卵母细胞中mRNA的差异,由此克隆到四个差异表达基因的全长cDNA,并对其进行了特征分析。第一个基因为未知基因CB102,它在彩鲫卵母细胞中的mRNA表达量远大于银鲫,预测它是一个跨膜蛋白。该基因为卵巢特异,在各个发育期的卵母细胞中都表达;在从受精后到桑椹期的胚胎发育早期也有高表达,然后表达量下降,到孵化期后检测不到其mRNA。第二个基因为新基因YA2,它在银鲫卵母细胞中特异表达,仅编码66个氨基酸,从受精后到胚都有高表达,表达量从尾芽期开始下降,在出苗期之后检测不到其mRNA。第三个基因为组蛋白H2A,其在银鲫和彩鲫间的差异主要表现在C末端序列。第四个基因为周期蛋白cyclin A2,其 cDNA全序列在鱼类中为首次报导。比较银鲫和彩鲫三种周期蛋白,其氨基酸序列的差异A2大于A1和B,3'非编码区的差异A2和B大于A1。在完全长成卵母细胞中,银鲫cyclin A2的mRNA表达量远高于彩鲫(≥20倍),cyclin A1与彩鲫相差不大(≈2倍),cyclin B则相同;cyclin A2的蛋白量也是银鲫中高,在两性生殖的彩鲫、红鲤和镜鲤中很低。在激素诱导卵母细胞成熟过程中,cyclin A2蛋白在银鲫中一直维持在高水平,在彩鲫中的表达量约在减数分裂中期I开始升高,在减数分裂中期II之前迅速下降。在完全长成的卵母细胞中,彩鲫中cyclin A1蛋白的高表达,而在银鲫中却检测不到cyclin A1蛋白,这与它们的cyclin A2 mRNA表达量相近不符。通过对这四个基因的分析和讨论,提出了一个解释雌核发育银鲫第一次减数分裂被抑制的假设。
Other AbstractIn this dissertation, the differentially expressed genes in fully grown oocytes between gynogenetic silver crucian carp (Carassius auratus gibelio) (gyno-carp) and gonochoristic crucian carp (Carassius auratus color variety) (gono-carp) were compared using subtractive hybridization method. Four differentially expressed full-length cDNAs have been cloned and their characterizations have been analyzed. The first one is the unknown gene CB102 whose transcripts are more abundant in fully grown oocyte of gono-carp. Sequence predication shows that it may be a transmembrane protein. It is an ovary specific gene and is expressed in all stages of oogenesis. In early embryogenesis, CB102 is expressed at high level from fertilization to morula stage. After that, its expression decreases and its mRNA can not be detected after hatching embryo stage. The second one is the novel gene YA2 that is specifically expressed in fully grown oocyte of gyno-carp and encodes a short peptide of 66aa. Its expression remains at high level from fertilization to neurula stage and decreases after tail bud stage, and its mRNA can not be detected after larvae stage. The third one is histone H2A. The sequences of histone H2A of the gyno-carp and gono-carp are quite different at C-terminal. The fourth gene is cyclin A2 whose full-length cDNA is reported in fish for the first time. Comparing the three cyclins' sequence differences between gyno-carp and gono-carp demonstrates that the amino acid sequences of cyclin A2 are more variable than cyclins A1 and B, and 3' untranslated region sequences of cyclins A2 and B are more variable than cyclin A1. In fully grown oocyte of gyno-carp, cyclin A2 mRNA is more than 20 folds, and cyclin A1 mRNA is about 2 folds comparing to that of gono-carp. However, cyclin B does not have such a difference. Western blotting analysis also demonstrates that a large amount of cyclin A2 proteins are stockpiled in fully grown oocyte of gyno-carp, while the protein is under the detectable level in fully grown oocytes of gonochoristic color crucian carp, red carp and mirror carp. During oocyte maturation induced by hormone injection, cyclin A2 protein in oocyte of the gyno-carp remains at a relative high level throughout the whole maturation division. As for the gono-carp, cyclin A2 protein is up-regulated when the oocyte enters metaphase I and is down-regulated before the oocyte enters metaphase II. Western blotting analysis reveals that gono-carp stockpiles a large amount of cyclin A1 proteins in its fully grown oocyte, while the protein is under the detectable level in gyno-carp, which is not accordant with the result of Northern blotting. Through the discussions and analysis on the four genes, a hypothesis has been postulated on the meiosis I inhibition of the gynogenetic silver crucian carp.
Document Type学位论文
Recommended Citation
GB/T 7714
谢京. 银鲫和彩鲫卵母细胞中差异表达基因的克隆及其特征分析[D]. 中国科学院水生生物研究所. 中国科学院水生生物研究所,2001.
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