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题名: 人工和天然雌核发育鱼类的生化遗传学研究
作者: 杨书婷
答辩日期: 2000
导师: 桂建芳
专业: 水生生物学
授予单位: 中国科学院水生生物研究所
授予地点: 中国科学院水生生物研究所
学位: 博士
关键词: 雌核发育 ; 生化遗传标记 ; 精子蛋白 ; 卵壳蛋白 ; 卵子抽提液 ; 蛋白纯化 ; 凝集素 ; 基因克隆
摘要: 本研究在了解和综述人工和天然雌核发育鱼类及其相关研究的现状和进展的基础上,以所在实验室在人工雌核发育鲢鱼(Hypophthalmichthys molitrix)和天然雌核发育银鲫(Carassius auratus gibelio)研究方面的前期工作为起点,主要利用生物化学方法开展了人工和天然雌核发育鱼类的遗传标记鉴定和发育调控机制问题等研究。研究内容主要包括两个方面。一是选择同工酶为分子标记指标,对两个人工雌核发育二倍体鲢鱼群体进行了遗传相似性和多态性比较分析,从生化遗传角度考察了各个雌核发育鲢鱼群体之间的遗传多样性,进而评价了人工雌核发育操作技术对鲢鱼群体遗传结构的影响。结果表明,各个雌核发育鲢鱼群体内不同个体间的酶谱表现出很大程度的一致性,具较高的纯合度,而不同雌核发育群体之间表现有明显差异。特别是肝脏、肌肉和血细胞中的酯酶谱带以其稳定的差异,建议作为区分不同人工雌核发育鲢鱼群体的生化遗传标记。二是以雌核发育银鲫的两性亲缘种彩鲫(Carassius auratuscolor variety)作为对照,从生化分析入手,对银鲫精子、卵壳以及卵子提取物等进行了分级分离等研究。首先,通过分级抽提得到精液的不同组份精浆、精头的膜、鞭毛和脱膜精头等,然后经不同的凝胶电泳系统,比较银鲫精子和彩鲫精子相应组份可溶性蛋白成分的差异。研究表明,经分级抽提的银鲫精子和彩鲫精了的各个组份都含有其特定的蛋白谱带。两种鱼精头膜的可溶性蛋白在SDS-PAGE电泳图谱上基本一致,而在非变性聚丙烯酰胺凝胶电泳图谱上则具有各自的特征性谱带。鞭毛可溶性蛋白的SDS-PAGE分析在雌核发育银鲫中揭示出一条特异的蛋白带。脱膜精头的可溶性蛋白在SDS-PAGE电泳图谱上差异明显,存在几条特征性蛋白带,并经Acid-Urea PAGE系统分析表明,这些特征性蛋白为碱性蛋白。接着,以雌核发育银鲫和两性生殖彩鲫的成熟卵为材料,分离卵壳,经处理得到卵壳可溶性蛋白组分。SDD-PAGE梯度凝胶电泳分析揭示出了雌核发育银鲫的特征性蛋白谱带。最后,借鉴非细胞体系(cell-free systems)的研究方法,制备了雌核发育银鲫和两性生殖彩鲫成熟卵子的抽提液,采用不同电泳系统对天然雌核发育银鲫和两性融合生殖彩鲫卵子抽提液中的蛋白成分进行比较分析。结果表明,虽然抽提液中蛋白质成分多而复杂,在SDS-PAGE胶上分辨不出相互之间的差异,但在非变性梯度凝聚电泳图谱上则各存在有一组差异的蛋白谱带,银鲫差异蛋白谱带的迁移率快于彩鲫差异蛋白谱带。后经P11和DE52离子交换柱以及CHT亲和柱层析等步骤,纯化得到雌核发育银鲫的差异蛋白。该蛋白经SDS-PAGE胶电泳和考马斯亮蓝显色表现为一条带,在经2D-PAGE胶电泳也表现为一个单点,表明该蛋白已被纯化,其分子量约为22 kDa。用该蛋白制备的多抗所进行的Western印迹也进一步证明该蛋白为雌核发育银鲫所特有。同时,经热变性处理研究表明,卵子抽提液经85 ℃变性处理20 min,其绝大部分蛋白被变性沉淀,上清液经55%(NH_4)_2SO_4饱和沉淀后,可分离到热稳定蛋白。该热稳定蛋白经12%SDS-PAGE胶电泳分离,呈现出分子量分别大于100 kDa和约29 kDa的两条蛋白带。经同等条件下获得的电泳图谱和Western印迹分析表明,从卵子抽提液中纯化的银鲫差异蛋白和热稳定蛋白为两种含量较高而又不同的蛋白因子。生化分析进一步表明,银鲫差异蛋白既不是糖蛋白,也不是磷蛋白,同时脂类蛋白显色也为阴性。雌核发育银鲫差异蛋白经氨基酸测序确定了其12个氨基酸肽段序列,用据此设计的引物,从银鲫卵母细胞cDNA文库中克隆出了其全长cDNA,并从彩鲫卵母细胞cDNA文库中也克隆出了与该差异蛋白基因同源的全长cDNA。通过同源基因查询和结构预测,确定它们均属于C型凝集素(lectin)蛋白家族中的成员,同时揭示出可能与雌核发育调控相关的一些差异特征,如银鲫的差异蛋白比彩鲫的同源蛋白少一个跨膜区。Western印迹和RT-PCR分析进一步揭示银鲫的差异蛋白在卵母细胞中特异表达,并经免疫荧光定位初步将其定位于卵膜上。
英文摘要: Genetic markers and gynogenetic mechanism were studied in artificial gynogenetic silver carp (Hypophthalmichthys molitrix) and natural gynogenetic silver crucian carp (Carassius auratus gibelio) by biochemical techniques on the basis of research status and previous achievements. There are two main parts included in this dissertation. First, two artificial gynogenetic populations that were obtained from different broodstocks of silver carp, Hypophthalmichthys molitrix, originated from two fishery farms in Wuhan during 1997, were analyzed by isozyme electrophoretic patterns. Genetic heterogeneity between different gynogenetic populations and genetic similarity within gynogenetic population were revealed, and genetic impacts on the artificially gynogenetic manipulation were assessed by analysis of population structures. In comparison with common hatchery silver carps, there were more similar isozyme electrophoretic patterns among the individuals of each artificially gynogenetic population, and therefore, there existed higher homogeneity because of artificial gynogenesis. Additionally, obvious differences between different gynogenetic populations were found. Some bands specific to each gynogenetic population were observed in EST isozyme zymograms from liver, muscle and red blood cells. Therefore, the specific bands would be suggested as biochemical genetic markers for discriminating these artificial gynogenetic populations. In order to reveal molecular mechanism responding natural gynogenesis in silver crucian carp, a series of biochemical and molecular studies had been initiated in the current study. We first performed the comparative studies on fractionation of sperm proteins between males from gynogenetic and gonochoristic crucian carp. Sperms from gynogenetic silver crucian carp and the related species of gonochoristic color crucian carp (Carassius auratus color varity) were extracted respectively by same methods at same conditions, and various fractions were collected. The fractions include sperm plasma, sperm head membrane, flagella, and demembranated sperm head. The soluble protein components of these extracted fractions and the whole sperm were analyzed by means of different polyacrylamide gel electrophoresis systems, such as nondenaturing polyacrylamide gel electrophoresis (ND-PAGE), SDS-PAGE and Acid-Urea PAGE system. The results showed that each fraction contains specific soluble protein components, and a few of soluble proteins are different in some fractions between gynogenetic silver crucian carp and gonochoristic color crucian carp. In the flagella fraction, one soluble protein component specific for gynogenetic silver crucain carp was observed by SDS-PAGE system. The soluble protein components in sperm head membrane fraction were analyzed by SDS-PAGE system and nondenaturing PAGE system, and found that they have same pattern in SDS-PAGE system but various patterns in nondenaturing PAGE system. It was suggested that the difference might be related to complex and functional status of the head membrane proteins. The significant difference of the soluble protein components between two types of fishes was discovered in demembranated sperm head fraction. Several proteins bands specific for gynogenetic silver crucain carp were revealed by SDS-PAGE analysis. Furthermore, the specific proteins were demonstrated to be basic proteins by Acid-Urea PAGE system. Then, the egg chorions were isolated from unfertilized and fertilized eggs of gynogenetic silver crucian carp and gonochoristic color crucian carp by homogenization and further purification techniques, and these preparations were analyzed by gradient SDS-PAGE. With basic resemblance, some differential protein bands were observed. Finally, a differential protein was identified from the gynogenetic silver crucian carp by comparing protein components in the egg extracts between the gynogenetic silver crucian carp and the closely related gonochoristic color crucian carp. When the extracts from silver crucian carp and color crucian carp eggs were subjected to SDS-PAGE, no differential protein bands were observed because of numerous protein components in the eggs. However, when the extracts were subjected to gradient ND-PAGE, a set of differential bands for the silver crucian carp was detected. Subsequently, a differential protein was purified to homogeneity by a series of chromatography steps, and by electrophoretic detection of SDS-PAGE and two-dimensional PAGE. Molecular weight of the purified protein was estimated to be about 22 kDa by comparing mobility of the protein with molecular weight standards on SDS-PAGE. Polyclonal antiserum against the purified protein was prepared by immunizing rabbit. Immuno-blotting analysis revealed a strong reaction band corresponding to the protein in extracts from silver crucian carp eggs, but not in that from color crucian carp eggs or other tissues. These results allowed us to suggest that this protein should be a specific component for silver crucian carp and might exist only in silver crucian carp ovary. Furthermore, this protein has been demonstrated to express at very early stages of oogenesis. At meantime, we obtained heat stable proteins from egg extracts of silver crucian carp and color crucian carp by heated the extracts at 85 ℃ for 20 min. Two bands were revealed after separation on 12% SDS-PAGE electrophoresis, one band is more than 100 kDa, the other is about 29 kDa. Both results of electrophoresis and Western blot reaction suggested that the purified differential protein should be different from the heat stable proteins, although the two kinds of proteins are all high contents in the egg extracts. Furthermore, the differential protein had been characterized as a non-glycoprotein without lipid and phosphors, because it stained negative for phosphorus, for lipid and also for carbohydrate. Partial sequences of 12 amino acids were determined from the purified differential protein, and a primer was designed based on the known amino acid sequences. Using PCR cloning method established before, we cloned the full-length cDNA of the differential protein from the oocyte eDNA library of silver crucian carp. And then, full-length homologous cDNA was also cloned from the oocyte cDNA library of color crucian carp. GeneBank database searches show that both of the predicted proteins belong to the C-type lectin family, and reveal some differential characterizations that may be related to the regulative mechanism on gynogenesis. For example, there is only one transmembrane region in the deduced protein of silver crucian carp, while there are two transmembrane regions in deduced homologous protein of color crucian carp. Furthermore, Western blot and RT-PCR results showed that the differential protein of silver crucian carp specifically expressed in oocytes, and it was preliminarily located on the oocyte plasma membrane by fluorescence immune assay.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.ihb.ac.cn/handle/342005/12534
Appears in Collections:中科院水生所知识产出(2009年前)_学位论文

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人工和天然雌核发育鱼类的生化遗传学研究.杨书婷[d].中国科学院水生生物研究所,2000.20-25
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