【作者】 廖梅杰；

【导师】 杨官品； 戴继勋；

【作者基本信息】 中国海洋大学 ， 海洋生物学， 2007， 博士

【摘要】 鲢（Hypophthalmichthys molitrix）和鳙鱼（Aristichthys nobilis）是中国特有的四大家鱼中的2个重要成员,主要分布在黑龙江、长江和珠江水系。传统人工养殖依靠天然鱼苗。但是,人口增长和人类经济活动使其天然产卵和孵化场消失或遭到破坏。人工育苗技术虽然解决了鱼苗的供应问题,但是不完善的遗传资源管理和使用方法,使这两种鱼的生长表现、抗病抗逆性和遗传多样性都明显降低。另外,因洪水导致的养殖个体逃逸和大规模人工鱼苗放流实践也使天然群体的遗传多样性受到干扰。对这些问题的研究迫切需要一套适用的分子标记。到目前为止还没有这两种鱼的微卫星DNA遗传图谱。蛋白感染因子（Proteinaceous infectious agents, prions）,也称朊病毒,是蛋白本质、导致传染性海绵样脑病（transmissible spongiform encephalopathies, TSE）的致病因子,一般指感染因子蛋白（prion protein, PrP）的凝聚体。养殖鱼类也可能通过饲料蛋白形成鱼类prion并引发鱼类TSE。本文以鲢和鳙鱼为研究材料,分离鲢微卫星DNA分子标记、研究鲢鱼的遗传多样性和鲢微卫星DNA标记在鳙鱼中的通用性、构建了鲢、鳙鱼的遗传连锁图谱,另外,还对鳙鱼的蛋白感染因子相关基因进行了克隆与分析,主要结果如下:利用FIASCO的方法构建了鲢（GT）n的微卫星DNA富集文库,检测结果证明该微卫星DNA文库的富集效率在35%左右。测序得到81条含有微卫星DNA的序列的克隆,共得到96个微卫星DNA位点。其中完美的微卫星DNA共68个,占71%;不完美的微卫星DNA共22个,占23%;复合的微卫星DNA有6个,占6%。在这些微卫星DNA位点中,重复单元（CA/GT）n有88个,占92%,另外我们还观察到A7、（AG/TC）n、（AT）12、（ATC）8、（TCCA）6等重复序列的微卫星DNA。所得到的微卫星DNA的重复次数在5-103次之间,大部分为10-20次重复,平均重复次数为14.5次。根据分离所得的鲢微卫星DNA位点,设计了67对微卫星DNA标记引物,有44对能够得到特异的扩增产物。利用这些引物对长江流域野生鲢进行遗传多样性分析,结果显示,44个微卫星DNA位点中,有40个为多态性的位点,共得到297个等位形式,各位点分别扩增得到的2-16个等位基因,平均每一个位点的等位基因数目为7.4个。40个多态性位点中,期望杂合度范围为0.07～0.91,平均的期望杂合度为0.69。这些位点显示出较高的多态性。利用这些引物对鳙鱼进行跨物种扩增试验,发现这些引物在鲢和鳙鱼中是通用的,均可以的到扩增产物。利用拟杂交策略和微卫星DNA和AFLP分子标记,以鲢、鳙鱼杂交亲本及其F1群体为作图群体,应用JoinMap3.0^?,构建了鲢（♂）和鳙鱼（♀）的中等密度遗传连锁图谱。在鲢（♂）图谱中,包含27个连锁群共271个标记（48个SSR和223个AFLP）,总图距为952.2cM,图谱覆盖率为82.8%。鳙鱼（♀）图谱包含30个连锁群共153个标记（27个SSR和126个AFLP）,总图距为852.0cM,图谱覆盖率为80.5%。共有18个微卫星DNA位点为两图谱共有。利用RT-PCR的方法,从同一个鳙鱼个体脑组织中克隆得到两种形式的蛋白感染因子基因序列。推测的蛋白感染因子蛋白具有明显的PrP结构特点,PrP-1蛋白含有526个氨基酸,分子量约为55kD,pI约为8.9;PrP-2蛋白含有519个氨基酸,分子量约为54.6kD,pI约为9.0。与斑马鱼3种形式的PrP相比较对照,这两种PrP蛋白属于PrP-2型。分子系统学分析显示,在疏水区域淡水鱼之间的同源性较淡水鱼与海水之间的同源性高,淡水鱼与海水鱼的PrP在结构上明显的不同主要表现在重复区域上出现重复肽段的重复次数的减少,据推测,重复片段的功能可能与盐代谢具有相关。更多还原

【Abstract】 Silver carp （Hypophthalmichthys molitrix） and bighead carp （Aristichthys nobilis） are two of the four most important pond-cultured fish species which inhabit the major river basins of China,including Heilongjiang River, Yangtze River and Pearl River.With the increase of human population and the intensification of economical activity,natural spawning and hatching grounds of the two fish are either polluted or altered,accelerating the loss of genetic resource and the decrease of natural fry production accordingly.Due to inappropriate broodstock management,continuous artificial propagation over a long period of time has caused tremendous decrease of genetic diversity and degeneration of disease resistance and growth performance. A set of appropriate markers are needed to detect these problems. Microsatellite DNA genetic linkage map of the two fish have not been constructed until now.Proteinaceous infectious agents （prions）, the aggregation of prion protein （PrP）, are the causing agents of transmissible spongiform encephalopathies （ TSE ）. PrP is highly diverse and has a wide range of distribution. It is highly possible that prion and TSE could form in cultured fish through feed.In this study, microsatellite DNA markers were isolated from silver carp and used to detect genetic diversity of silver carp and cross-species amplification in bighead carp. Genetic linkage map of the two fish were constructed. Prion protein encoding gene of bighead carp was cloned and characterized from bighead carp.The following results were received: A （GT）n enriched library was constructed for silver carp using the FIASCO method. Of the 81 sequenced clones, 96 microsatellite DNA were identified,among them 68 were perfect, 22 were imperfect and 2 were compound. Besides 88 （CA/GT）n microsatellite DNA, other types of microsatellite DNA such as A7、（AG/TC）_n、（AT）₁₂、（ATC）₈、（TCCA）₆ were also been observed. The repeat number ranged from 5 to 103 with an average of 14.5.Sixty-seven pairs of silver carp microsatellite DNA markers were designed. Of them, 44 could get good amplification. All of the 44 microsatellite DNA markers used to investigate polymorphisms of 41 wild silver carps and seven wild bighead carps collected from Yangtze River. In silver carp, 40 markers were polymorphic. A total of 297 alleles were detected at 40 polymorphic loci. The number of alleles per locus varied from two to 16 with an average of 7.4 and the expected heterozygosities of these loci ranged from 0.07 to 0.91 with an average of 0.69. All markers amplified both silver carp and bighead carp DNA.Genetic maps of silver carp and bighead carp were constructed using microsatellite DNA （or simple sequence repeat, SSR） and amplified fragment length polymorphisms （AFLP） markers and two-way pseudo-testcross strategy. The map of silver carp consisted of 271 markers （48 SSR and 223 AFLP） which were assembled into 27 linkage groups, of which 22 contained at least 4 markers. The total length of silver carp map was 952.2 cM, covering 82.8% of the estimated genome size. The map of bighead carp consisted of 153 markers （27 SSR and 126 AFLP） which were organized into 30 linkage groups, of which 19 contained at least 4 markers. The total length of bighead map was 852.0 cM, covering 70.5% of the estimated genome size. Eighteen SSR markers were common to both maps. Two transcripts of bighead carp prion protein encoding gene were cloned and characterized through RT-PCR approach. The deduced amino acid sequences held conservative structure features of prions. One form of bighead carp PrP consisted of 526 residues with an estimated molecular weight of about 55kD, and the pI was about 8.9. The other one consisted of 519 residues with an estimated molecular weight of about 54.6kD,and the pI was about 9.0. They belonged to PrP2 when compared with 3 forms of PrP of Zebrafish. Phylogenetic analysis demonstrated that the homology between freshwater fish is higher than that between freshwater fish and marine fish. The number of tandem repeats decreased in freshwater fish, so it was speculated that the function of PrpC is relevant to salt metabolism.更多还原