【作者】 张婷；

【导师】 祝茜；

【作者基本信息】 山东大学 ， 海洋生物学， 2010， 硕士

【摘要】 辽东湾斑海豹(Phoca largha)是惟一一种能在我国海域繁殖的鳍足类动物,在《国家重点保护野生动物名录》中被列为国家Ⅱ级保护动物,其分布区域位于世界上8个繁殖区的最南端,已经与其他繁殖区域的斑海豹产生一定程度的隔离。辽东湾斑海豹数量已不足千头,环境污染、栖息地的破坏和猎捕等加剧了其自然种群数量的下降,种群状况已岌岌可危。虽然对辽东湾斑海豹开展了一定的研究和保护工作,但主要集中在形态结构、习性、分布特征以及人工饲养和繁殖上,大多数保护活动仍然是在缺乏保护遗传学研究的情况下进行的。因此,通过以往的研究不能准确全面的获得其保护遗传学信息。主要组织相容性复合体(major histocompatibility complex, MHC)是具高度多样性的核基因,在阐明种群间的变异和适应机制方面更具优越性。通过MHC基因的变异分析,可有效评估种群遗传多样性水平。本文以辽东湾25份斑海豹样本为研究对象,分析了MHC-DQB第二外显子的基因多态性,探讨和评价了其种群遗传多样性水平,及在抵抗疾病和适应环境变化方面的能力和潜在威胁。从25份斑海豹样本的270多个克隆中,得到141bp的片段。在这些序列中发现21个变异位点,定义了12个等位基因。氨基酸变异率为25.5%。等位基因之间的遗传距离范围是0.0071～0.1064,平均值为0.0577,不同等位基因之间的碱基差异是1～15bp,平均差异数为8bp,表现出较丰富的遗传多样性。在56条序列中发现13种可疑序列,怀疑是非特异性扩增造成的微卫星序列。对核苷酸替代的类型及位点进行分析,发现非同义替换率明显高于同义替换率(平均dN/ds=2.07),由此造成的氨基酸替换集中在肽结合位点PBR附近,表明斑海豹的DQB基因受到强烈的正选择或平衡选择作用。大部分替换的氨基酸和被替换的氨基酸理化性质不同,这种理化性质的改变,可能与它们识别和提呈不同的抗原有关。构建的系统发育树及TCS网络图显示,鳍足类MHC基因表现出跨物种进化现象,ds值分析也支持这一结论。斑海豹DQB等位基因分布不平衡,稀有等位基因不成比例,暗示辽东湾斑海豹可能经历过遗传瓶颈效应。11个样本出现多于两条等位基因的情况,推测存在基因重复现象。DQB第二外显子包含两个富含GC的区域(即GCi和GCii),含类似X的序列,显示出一种纯粹的重组信号。根据系统发育树、中接网络图、TCS网络图和种群遗传结构分析对斑海豹的位点进行讨论,认为斑海豹的59个等位基因可能来自DQB的两个位点和DRB的一个位点。通过计算各个位点的单倍型多样性、核苷酸多样性和平均核苷酸歧义度等遗传多样性参数,并与其他鳍足类的遗传距离、每个个体所含等位基因数、碱基及氨基酸变异等参数进行比较,发现各个位点的遗传多样性水平较高。通过Tajima’D检验和Fu’Fs检验,以及对非同义替换率与同义替换率进行比较,结果显示,DQB受到正选择的作用。这在一定程度上支持了达尔文的自然选择学说。基因重复和每个位点的基因多样性共同作用,使斑海豹MHC表现出丰富的多态性,可以最大限度的适应多变的病原体环境。但是,等位基因分布不平衡,如果稀有等位基因在遗传漂变的作用下在斑海豹种群中消失,将会导致斑海豹多样性的进一步丧失,从而丧失对多样化病原体的抵抗能力。因此,在保护斑海豹生存环境的同时,还必须尽可能多地保护物种的遗传多样性,才能真正达到保护物种的目的。更多还原

【Abstract】 The largha seal Phoca largha distributing in Liaodong Bay is the only pinniped that breeds in coastal areas of China. It has been listed in Order II under the Wild Animal Protection Law in China. Liaodong Bay is the southern-most end of the eight breeding areas of the largha seals, where the population has few or no genetic exchanges with the ones from the other breeding areas. During to the pollution of coastal waters, loss of habitats, and poaching, the population in Liaodong Bay is estimated to be less than 1000 individuals. The previous studies mainly focus on its morphology, habits, distribution and artificial feeding and breeding, but the conservation is still undertaking without the information on genetic researches.The major histocompatibility complex (MHC) is one of the nuclear genes with high degree of diversity, and is significantly useful to clarify the mechanisms of mutation and adaption among populations. The level of genetic diversity can be effectively assessed by the mutation analysis of MHC gene. Therefore, the gene polymorphism of the second exon of MHC-DQB loci obtaining from twenty-five specimens, and the abilities and potential threats in resisting diseases and adapting environment change within this population, was studied.141bp nucleotide sequences were extracted from more than 270 clones of the twenty-five largha seals. From the sequences, twenty-one variable sites were found and twelve alleles were defined. The variability was 25.5% at the amino acid level. The nucleotide diversity was from 0.0071 to 0.1064 (average 0.0577), and the base difference was from lbp to 15bp (average 8bp). All these results showed a relatively high level of genetic diversity in this population. Fifty-six questionable sequences with thirteen different length were found, considered to be microsatellite loci caused by non-specific amplification.High rate of non-synonymous vs synonymous (dN/ds=2.07) substitution especially in the peptide-binding region (PBR) suggested strong positive selection or balancing selection for maintaining high MHC diversity at the DQB loci. Most physical and chemical properties of the new amino acids translated after nucleotide substitutions were different from those of the original ones, which was interpreted as being relative with identifying and presenting different antigens.Phylogenetic dendrogram and TCS network indicated trans-species evolution in the pinniped MHC gene, which was also supported by the ds-value analysis. The imbalanced distribution of DQB alleles and the disproportion of the rare alleles suggested the genetic bottleneck effect in the history.Eleven individuals holding more than two alleles suggested duplication of DQB gene. The second exon of DQB contained two GC-rich regions (GCi and GCii) and one X-like sequence, showing a pure signal of gene recombination. Based on the analyses from the phylogenetic dendrogram, median-joining network, TCS network, and genetic structure analysis, it concluded that the fifty-nine alleles of the largha seals may be from two DQB loci and one DRB locus. High level of genetic diversity in every locus was proposed from the parameters of haplotype diversity, nucleotide diversity and average number of nucleotide differences, as well as from comparisons with other pinniped’s parameters, viz. pairwise distance, number of alleles in each individual, mutations of the nucleotide and amino acid. The results of Fu’Fs and Tajima’D test, and the comparison between rates of non-synonymous and synonymous substitutions, showed positive selection on DQB, which supported Darwin’s theory of natural selection to some extent.Gene duplication and genetic diversity per locus work together to make the MHC of the largha seal showing polymorphism, which could adapt sufficiently to the changeable pathogen environments. However, the imbalanced distribution of alleles and the possibility of losing rare alleles in genetic drift would further lead to the loss of the diversity among the populations of the largha seal and the abilities to resist different pathogens in the future. Therefore, the conservation of largha seals should not only focus on their living environments, but also their genetic diversity.更多还原