【作者】 成述儒；

【导师】 罗玉柱；

【作者基本信息】 甘肃农业大学 ， 动物遗传育种与繁殖， 2010， 博士

【摘要】 为了研究藏绵羊DQA1、DQA2和DRB3基因多态性,确定其等位基因数、核苷酸多态位点、氨基酸多态位点、各等位基因单倍型连锁,遗传关系和进化意义、以及DQA1、DQA2和DRB3基因作为分子遗传标记的可能性。研究采用PCR-SSCP方法检测了900只藏绵羊DQA1、DQA2和DRB3基因外显子2的等位基因,并克隆、测序群体内变异产生的各等位基因序列,就等位基因单倍型序列数据开展分析。主要研究结果如下：1.藏绵羊DQA1、DQA2和DRB3基因外显子2的PCR-SSCP检测,克隆测序和序列对比分析,研究共获得17个DQA1等位基因(包括缺失的一种),16个DQA2等位基因和51个DRB3等位基因。其中5个DQA1等位基因,8个DQA2等位基因和50个DRB3等位基因是本研究首次发现的新等位基因。藏绵羊中DQA1、DQA2和DRB3基因新等位基因的出现可能与其所处的地理环境和气候条件与国外品种不同有关。2.900只藏绵羊中有218(24.22%)只个体没有检测到DQA1基因,属于DQA1基因缺失的类型。藏绵羊16个DQA1外显子2等位基因(缺失除外)序列共发现56个核苷酸多态位点,藏绵羊16个DQA2外显子2等位基因序列共发现71个核苷酸多态位点,藏绵羊51个DRB3外显子2等位基因序列共发现91个核苷酸多态位点。研究表明藏绵羊DQA1、DQA2和DRB3基因均具有较高的多态性,这与藏绵羊常年生活在恶劣环境下(如高寒)所具有的适应性强、耐粗放管理、抗病性强的特点相一致。在研究的3个基因中DRB3基因多态性最丰富,DQA2和DQA1次之。3.藏绵羊16个DQA1外显子2等位基因共发现27个氨基酸多态位点,16个DQA2外显子2等位基因共发现40个氨基酸多态位点,51个DRB3外显子2等位基因共发现50个氨基酸多态位点。4.系统聚类分析表明藏绵羊DQA1和DQA2基因均呈两支分化,表明藏绵羊DQA1和DQA2基因最初可能是由2个等位基因分化成两大类等位基因的。而DRB3基因呈3支分化,表明DRB3基因最初可能是由3个等位基因分化成3大类等位基因的,也可能第Ⅲ支的产生是由第Ⅰ和第Ⅱ支突变衍化而来。5.研究发现的16个DQA2等位基因中,等位基因*N,*K和*F属于类-DQA2基因,在类-DQA2基因存在的个体上均未检测到DQA1基因,表明在DQA1基因缺失时,类DQA2基因可能占据了DQA1基因座位,同时证明绵羊DQA2基因在MHC上存在复制体。6.藏绵羊DQA1、DQA2和DRB3基因与牛的DQA1、DQA2和DRB3基因都具有较高的同源性,预示绵羊和牛的DQA1、DQA2和DRB3基因最早可能来源于它们分歧以前的共同祖先的原始序列；DQA1、DQA2和DRB3基因在与其相关的特定抗原刺激下发生的免疫应答反应在绵羊和牛上可能具有相似性。7.研究发现了35种"DQA1-DQA2"单倍型,78种"DQA2-DRB3"单倍型,105种"DQA1-DQA2-DRB3"单倍型。发现的单倍型数均低于理论值,而且单倍型频率分布极不平衡,表明DQA1、DQA2和DRB3基因间存在单倍型连锁不平衡现象。研究首次发现藏绵羊MHC-DQ亚区的DQA1和DQA2基因与其MHC-DR亚区的DRB3基因存在单倍型连锁遗传现象。8.绵羊DQA1、DQA2和DRB3基因可以作为分子遗传标记基因,在进行与绵羊DQA1、DQA2或DRB3相关的疾病抗性选择时,结合"DQA1-DQA2-DRB3"单倍型的选择,将会提高选择的可靠性。更多还原

【Abstract】 With the view of studying polymorphism of DQA1,DQA2 and DRB3 genes exon 2 in Tibetan sheep, determining the number of alleles, nucleotide polymorphism sites, amino acid polymorphism sites, haplotype recombination of alleles, genetic relationship among every allele and evolutionary significance, as well as the possibility of DQA1,DQA2 and DRB3 genes as the molecular genetic markers. Using PCR-SSCP method, we detected 900 Tibetan sheep DQA1,DQA2 and DRB3 gene exon 2 alleles at the first time, and cloning and sequencing every allele which comes from variation in population, then analyzing the haplotype sequence data of allele. We got the following conclusions:1.By PCR-SSCP detecting, cloning, sequencing and analysising of sequence comparison on DQA1,DQA2 and DRB3 gene in 900 Tibetan sheep,17 DQA1 alleles, 16 DQA2 alleles and 51 DRB3 alleles were obtained. There are 5 new DQA1 alleles,8 new DQA2 alleles and 50 new DRB3 alleles in them were found first time in this study. The appearance of new DQA1,new DQA2 and new DRB3 alleles in Tibetan sheep may be related to the difference of geographical surrounding and climatic conditions between the Tibetan sheep and exotic sheep breeds.2.There are 218 individuals (24.22%) that hadn’t detected DQA1 gene in 900 Tibetan sheep, belong to the DQA1 null haplotype. There are 56 nucleotide polymorphism sites were found in 16 DQA1 gene exon 2 alleles,71 nucleotide polymorphism sites in 16 DQA2 gene exon 2 alleles, and 91 nucleotide polymorphism sites in 51 DRB3 gene exon 2 alleles. The results showed that DQA1,DQA2 and DQA3 genes had high polymorphism, which probably attribute to the consistency of Tibetan sheep trait by living in wicked environment, high adaptability, extensive resistance, and high disease resistance. DRB3 gene has the richest polymorphism in the 3 MHC genes,DQA2 and DQA1 take the second place.3.There are 27 amino acid polymorphism sites were found in 16 DQA1 gene exon 2 alleles,40 amino acid polymorphism sites in 16 DQA2 gene exon 2 alleles, and 50 amino acid polymorphism sites in 51 DRB3 genes exon 2 alleles in Tibetan sheep.4.Phylogenetic analysis of the DQA1 and DQA2 gene exon 2 haplotypes in Tibetan sheep showed that two different clusters were found, which indicated that DQA1 and DQA2 gene may be developed into two main type alleles by 2 initially alleles.However, the DRB3 gene has 3 clusters, it showed that DRB3 gene were delveloped into three main type alleles from 3 initially alleles, or came from the first branch and second branch’s evolution or mutation.5.In 16 DQA2 alleles,*N,*K and *F alleles belong to the DQA2-like gene. There are not detected DQA1 genes in the individuals with DQA2-like gene. It showed that DQA2-like gene probable took up the locus of DQA1 gene when DQA1 gene was deletion, and DQA2 gene had duplication in MHC genes as well.6. DQA1,DQA2 and DRB3 genes in Tibetan sheep and cattle has high homology, it indicated that sheep and cattle DQA1,DQA2 and DRB3 gene may be derived from primordial sequences that were present in a common ancestor and have persisted in the sheep and cattle populations since their divergence, and the observation that sheep and cattle share similar allelic sequences may be evidence of the need for a specific immune response to a common pathogen.7.Thirty five types of "DQA1-DQA2" gene haplotypes, seventy eight types of "DQA2-DRB3" haplotypes and one hundred and five types of "DQA1-DQA2-DRB3" haplotypes were found in this study. The number of haplotypes is less than theoretical value, and haplotype frequency distribution is extremely imbalance, showed that the DQA1,DQA2 and DRB3 genes haplotypes have linkage disequilibrium phenomenon. It was the first time found that haplotype recombination of DQA1 and DQA2 genes in MHC-DQ subregion with DRB3 gene in MHC-DR subregion8.DQA1, DQA2 and DRB3 genes can be used as molecular genetic markers in sheep, during the selection of disease resistance related to DQA1,DQA2 or DRB3 gene, combined with "DQA1-DQA2-DRB3" haplotype choice, will enhance the selection reliability.更多还原