【作者】 徐成；

【导师】 张培军； 王可玲；

【作者基本信息】 中国科学院海洋研究所 ， 海洋生物学， 2000， 博士

【摘要】 本研究应用同工酶和随机扩增多态性DNA（RAPD）方法，分析了牙鲆野生种群和雌核发育群体的遗传多样性和遗传分化水平；应用RAPD方法对比分析了雌核发育牙鲆的亲鱼和子代DNA样品；采用PCR扩增牙鲆SRY同源片段并进行了序列分析；将RAPD扩增产物中性别连锁DNA片段克隆与测序。主要结果有：1.牙鲆雌核发育群体同工酶的Ldh-C，Cat两个基因座位发生了重组，基因座位 与着丝点之间的重组率分别为52.6％和29.8％；雌核发育群体RAPD分析发现136 个DNA片段中有22个发生基因分离，但由于研究方法的局限，不能确定是否 发生基因重组。2.牙鲆雌核发育群体的同工酶多态座位比例和平均杂合度分别为6.90％和 0.0350；研究中提出了RAPD遗传多样性参数修正算法；野生种群RAPD多态片 段比例37.57％，多态座位比例20.88％，平均杂合度0.0852；雌核发育群体RAPD 多态片段比例16.18％，多态座位比例8.98％，平均杂合度0.03898。同工酶与 RAPD方法得出结果基本一致。3.牙鲆雌核发育群体与野生群体之间的RAPD遗传相似度I=0.9036，遗传距离 D=0.1014，已超过一般鱼类地理种群之间的遗传距离值。雌核发育群体的遗 传多样性指数（H_o=7.1982）远低于野生群体（H_o=28.0986）。雌核发育 野生群体H_pop/H_sp=0.9716，(H_sp-H_pop)/H_sp=0.0284，表明97.16％的遗传多 样性是由群体内不同个体间的差异造成的，只有2.84％遗传多样性与群体间分 化有关。4.分析过同工酶多态座位比例、同工酶平均杂合度、RAPD多态片段比例、RAPD 多态座位比例、RAPD平均杂合度、群体遗传多样性指数H_o等遗传多样性参数， 牙鲆雌核发育群体各参数比野生群体减少55.39％-77.74％，说明它的遗传多 样性严重损失。5.采用RAPD对照分析亲本与雌核发育子代胚胎的DNA样品。结果表明，雌鱼有、 雄鱼无的基因，雌核发育子代表达：雌鱼无、雄鱼有的基因，雌核发育子代不 表达，正常受精二倍体对照组表达正常。证明牙鲫雌核发育子代的遗传物质来 自母本，雌核发育诱导使用的精子经过紫外线灭活后，遗传物质己被破坏，其 携带的遗传信息没有传给子代，在DNA水平证明了雌核发育诱导方法和结果的 可靠性。 6．进行了牙怦性别决定机制研究。PCR分析了哺乳动物性别决定基因SRY同源片 段在牙鲫的表达情况。扩增产物均无个体差异，也没有性别差异。将扩槽产物 中信号最强的sry七片段测序，其648bn序列与人SRY基因相应的419bn片段 进行了同源性分析，两者同源性为35％，同源性很低，只有随机的碱基重合。 由于牙鲫 SRY PCR扩增带没有全部分析，不能肯定牙鲫没有 SRY同源片段。RAPD 分析确定3个引物的4条扩增片段与性别相关，分别克隆、测序，5134和5145－S 两个片段得到了完整序列。更多还原

【Abstract】 Isozyme and Random Amplified Polymorphic DNA (RAPD) were employed to investigate the genetic diversity and genetic differentiation of wild and gynogencsis population in Paralichthys olivaceu. DNA samples of parent and filial generation of gynogenesis were comparatively analyzed by RAPD. The homologous fragment of SRY gene from flounder genome was PCR amplified and sequenced. Some sex-linkage bands in RAPD amplification were cloned into bacteria vector and sequenced. 1. Two isozyme loci, Ldh-C and Cat, were found to be recombined in their chromosomes in the gynogenesis population of Paralichthys olivaceu. The recombination rates between isozyme locus and centromere were 52.6% and 29.8% respectively. The high recombination rates may be related to the small size of fish chromosome arms. Total 22 out of 136 DNA bands amplified by RAPD were found segregation in gynogenesis. However, whether these bands caused by recombination were hard to perorate owing to the analysis method restriction. 2. Isozyme mean proportion of polymorphic loci (P) and the average heterozygosities (H) of gynogenesis population in Paralichthys ohvaceu were 6.90% and 0.03 50 respectively. In order to obtain correct results we developed a modified arithmetic of genetic diversity of RAPD. P and H of wild population by RAPD were 20.88% and 0.0852, proportion of polymorphic bands was 37.57%; P and H of gynogenesis population by RAPD were 8.98% and 0.03 898, proportion of polymorphic bands was 16.18%. Results from RAPD and isozymc analyses were shown to be accorded. 3. Genetic similarity and genetic distance between wild and gynogenesis population were 0.9036 and 0.10 14, respectively. Genetic distance was higher than average level between geographical populations of fish. The index of genetic diversity of gynogenesls vi population (H0 =7.1982) was lower than that of wild population (R. =28.0986) Genetic differentiation between gynogenesis and wild population, H,,O,/H. = 0.97 16, (H-H~) IHsp = 0.0284, showing 97.16% distributed within populations and only 2.84% between populations. 4. All parameters, including mean proportion of polymorphic loci (P) and the average heterozygosities (H) of isozyme; P and H as well as proportion of polymorphic bands of RAPD; index of genetic diversity (H0), indicated the genetic diversity of gynogenesis population badly lose comparing with that of wild population. The proportions of reduction were 55.390/c - 77.74%. 5. DNA samples of parent and filial generation of gynogenesis were comparatively analyzed by RAPD. Those DNA bands expressed in female parent but not in male parent also expressed in gynogenesis filial; DNA bands expressed in male parent but not in female parent didn抰 express in gynonensis, while normally expressed in dcploid control fish. Germ plasm of sperm had been destroyed thoroughly by UV radiation, all germ plasm of gynogenesis filial were from female parent. 6. To detect sex determination mechanism of Paralichthys olivaceu, the homologous fragment of SRY gene was PCR amplified and sequenced. PCR amplification had no difference between individual and sex. A 648bp fragment from Paralichihys olivaceu, named sry-2, was sequenced and only had 35% homologous compared with human SRY. Four out of 1533 RAPD amplification DNA bands were found to be sex linkage. These bands were then cloned into bacteria vector and sequen更多还原