【作者】 庞震国；

【导师】 常亚青；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 海洋生物学， 2009， 博士

【摘要】 第一部分刺参遗传连锁图谱的构建利用AFLP和微卫星标记以一个远缘地理杂交家系为作图群体,以拟测交理论为作图策略,构建了刺参Apostichopus japonicus的雌性和雄性遗传连锁图谱。利用62对AFLP引物组合,对亲本和93个个体进行了分离分析,共得到3572个标记,其中具有多态性的标记为点数为1133个,多态性比例为31.5%。平均每个引物对产生18.3个多态片段。1133个多态性位点中,294个标记在父母本中都出现并在后代中分离,839个标记在父本或母本中出现并在子代分离,其中母本443(52.8%)个分离标记,父本396(47.2%)个分离标记。卡方检验显示,556个标记符合孟德尔1:1分离。筛选微卫星标记中30个在家系中具有作图信息,其中19个可用于母本作图,19个可用于父本作图。利用Mapmaker 3.0/EXP软件对分离标记进行连锁分析。358个分子标记用于雌性遗传连锁分析,其中包括19个微卫星标记,199个AFLP标记和11个微卫星标记定位在雌性框架图谱中。雌性框架图谱共有25个连锁群,总长度为3148.3 cM。标记之间最大间隔为37.5 cM,平均间隔为17.0 cM,连锁群的分子标记数最大为21个最小为4个。用于雄性连锁分析的分子标记共有332个,其中包含19个微卫星标记。207个标记定位于23个连锁群上,其中AFLP标记数为196个,微卫星标记数为11个。23个连锁群总长度为3059.8 cM,标记间最大间隔38.6 cM,平均间隔16.6 cM。每个连锁群的长度范围在40 cM到271.4 cM之间,标记数在18到4个之间,包括二、三连体在内的雄性连锁图谱共覆盖3227 cM。雌雄基因组的预测长度分别为4053.7 cM和3816.3 cM,因此,所构建的雌雄连锁图谱覆盖率分别为84.0%和84.5%。通过共有微卫星标记,有3个连锁群在两个图谱中对应。分子标记在两个图谱中呈均匀分布,没有成簇现象。分子标记筛选、遗传图谱的构建为刺参分子标记辅助选择育种,经济性状QTL定位和比较基因作图打下基础,并且最终将促进中国刺参遗传改良和新品种的培育工作。第二部分卵母细胞成熟过程研究对刺参卵母细胞的体外诱导成熟方法进行探索,并且通过透射电子显微镜、扫描电子显微镜和激光共聚焦显微镜结合超薄切片技术及间接免疫荧光染色技术研究卵母细胞成熟的生物学过程。分别利用在海参中有诱导作用的海星神经提取液和二硫苏糖醇(DTT)对从成熟卵巢解剖出的刺参卵母细胞进行诱导成熟试验,结果显示,刺参的卵母细胞在海水中不会发生自发成熟,海星神经提取液以及神经提取液加滤泡悬浮液对卵母细胞没有诱导成熟的作用,而二硫苏糖醇处理可以诱导卵母细胞生发泡破裂,当DTT溶液的浓度处于10-1 mol/L到10-3 mol/L之间时,对刺参卵母细胞的促熟作用较为明显,在10-2 mol/L左右时,卵母细胞的诱导成熟率可以达到90%以上。未成熟的卵母细胞处于第一次减数分裂前期,不具备受精能力。经DTT诱导后,生发泡发生移动并破裂,染色体排列到赤道板上,然后第一、二极体先后排出,诱导成熟的卵母细胞排出极体的时间与自然受精的卵子一致。在卵母细胞成熟过程中有受精膜举起的现象,说明精子入卵不是受精膜举起的必要条件。卵母细胞只有在生发泡破裂之后才具有受精能力,从生发泡破裂到第二极体排出前的整个减数分裂过程中,卵母细胞都可以受精,但是只有在第一极体排出前受精的卵母细胞才能发生卵裂。人工促熟的卵母细胞受精后,形成的胚胎似乎并不能正常发育,试验过程中最多只得到了8细胞期的胚胎,之后细胞便产生畸形并停止分裂,最终裂解消失。利用DTT诱导刺参卵母细胞成熟,综合运用光镜、电子显微镜及激光共聚焦技术观察卵母细胞成熟过程,分析了卵母细胞表面动物极突起在卵母细胞脱滤泡中的作用,以及卵母细胞动物极突起和微管组织在生发泡移动过程中所起的作用。卵母细胞通过动物极突起连接到滤泡上,在脱滤泡作用开始时,卵母细胞动物极突起刺入并穿过滤泡膜,使得滤泡膜表面形成一个缺口,然后整个卵母细胞开始从缺口处挤出。脱滤泡的作用力来源有两种:胶膜层的水合作用以及滤泡的收缩。通过激光共聚焦显微镜观察,未成熟的卵母细胞中存在5种类型的微管,并且有两个微管组织中心锚定在卵母细胞突起下方。减数分裂重新启动时,生发泡沿微管向动物极移动,至细胞膜下时破裂。微管解聚对照实验证明驱动生发泡移动的细胞结构为微管束。生发泡破裂后,两个微管组织中心组织形成减数分裂纺锤体,然后第一极体以“收缩-排出”的独特方式排出。本研究填补了刺参繁殖生物学研究中的缺失环节,为刺参人工繁育技术提供理论基础和指导。更多还原

【Abstract】 Part I. Genetic linkage map constructiong of Apostichopus japonicusAmplified fragment length polymorphisms (AFLPs) and microsatellite markers were used for genotyping in sea cucumber Apostichopus japonicus. Sixty-two selected AFLP primer combinations produced 3572 fragments, of which 1133 (31.5%) were polymorphic in the mapping population. Each primer combination produced 18.3 polymorphic fragments on average. Among those 1133 polymorphic markers, 294 markers were from both female and male parents , 443 (52.8%) and 396(47.2%)markers were segregating through the female parent and the male parent respectively. Chi-square test indicated that 556 markers segregate in Mendelian ratio. 30 microsatellite loci screened were informative, 19 of them were heterozygous in the female and another 19 were heterozygous in the male parent.Mapmaker/EXP 3.0 software was used to process segregation analysis in the mapping population with 93 progenies. 358 markers were used in female segregation analysis, which involved 19 microsatellite markers. 199 AFLP markers and 11 microsatellite markers were anchored to the female linkage map. The female framework map is consisted of 25 linkage groups with the total length of 3148.3 cM. The max interval between markers is 37.5 cM and the average interval is 17.0 cM. The number of markers in each linkage group varies from 21 to 4. 332 markers were used in male segregation analysis. Among them 19 were microsatellite markers. 196 AFLP markers and 11 microsatellite markers were anchored in 23 linkage groups of the male framework map with the whole length of 3059.8c. Each of the linkage group contains from 4 to 18 markers and their average interval is 16.6 cM. The estimation of genome length of Apostichopus japonicus is 4053.7 cM for the female and 3816.3 cM for the male. The observe coverage was 84.0% for the female and 84.5% for the male.By using microsatillte makers heterozygous in both parents, three homologous pairs of linkage groups were identified. All markers were uniformly distributed without clustings. Some of the distorted markes were linked together.Molecular marker development and genetic linkage map construction provide the base for marker assisted selection, QTL mapping and comparative genome mapping. Part II. Research on oocyte maturation in Apostichopus japonicusThis research explored the methods of maturation induction of oocytes in vitro in sea cucumber Apostichopus japonicus. Using transmission electron microscopy (TEM), scanning electron microscopy (SEM) and laser scanning confocal microscope, as well as technique of ultra-thin slicing and indirect immunofluorescent staining, we study the biological process of oocyte maturation in A. japonicus.Nerve extraction substance (NES) of sea star and dithiothreitol (DTT) has been reported to be effectively to induce oocyte maturation in sea cucumbers. In this study we tested their effects on oocyte maturation induction in sea cucumber A. japonicus. The results are as follows, oocyte of A. japonicus does not get mature spontaneously. Neither NES alone nor NES with follicle suspension can induce oocyte maturation. While DTT can prominently increase the percentages of germinal vesicle break down (GVBD). When the concentration of DTT is between10-1 mol/L and 10-3 mol/L, the effect of maturation induction is more sufficient. When the concentration is about 10-2 mol/L, the percentage of GVBD can reach up to more than 90%. Immatured oocyte of A. japonicus is arrested in prophaseⅠand is not fertilizable. After being treated with DTT, the germinal vesicle (GV) migrates and breaks down and the chromosomes are rearranged to the metaphase plate, followed by extrusion of the first and second polar body. The polar body extrusion process of induced maturation is similar to naturally fertilized eggs. Rising of fertilization membrane during induced maturation indicates insemination is not necessary to form the fertilization membrane. The oocyte will not become fertilizable until GVBD has happened and can still be fertilized after the second polar body extrusion. However, only those were fertilized before first polar body extrusion could process cleavage, and the embryos could not develop normally.Using light microscopy, electron microscope and confocal scanning microscope, we observed the process of DTT induced oocyte maturation in sea cucumber A. japonicus and analyzed the role of animal process of oocyte during ovulation and oocyte maturation. What’s more, we studied the role of microtubules and microtubule organizing center (MTOC) upon GV migration. The oocyte of A. japonicus connects to the follicle via animal pole process. When placed in normal sea water, ovulation started spontaneously. The animal pole process punched in to the follicle and made a gap on it, and then the whole oocyte got out through this gap. Two forces may response to ovulation: the hydration of jelly space and contraction of follicle. Two MTOC are anchored beneath the animal pole process. Five types of microtubules were found in oocytes during maturation. After the onset of meiosis reinitiation, the GV stated to migrate along microtubules to the animal pole and attached to the site beneath oocyte membrane, that followed by GVBD. Oocytes treated with microtubule microtubule depolymerization agents before maturation induction failed to process GV migration. This result indicates microtubules are indispensable during GV migration. Afte GVBD, the two MTOCs begin to organize the meioses spindle. The animal pole process retracted back and the polar body extruded from the site of former animal pole process. This study fills up a gap in reproductive biology of A. japonicus.更多还原