【作者】 马长宾；

【导师】 孙延鸣；

【作者基本信息】 石河子大学 ， 临床兽医学， 2010， 硕士

【摘要】 野生盘羊是新疆优良的野生动物资源,该品种具有体型大、抗病力强、适应性好、产肉性能强和瘦肉率高等特点。为充分利用其优良的遗传资源,本实验室试图用野生盘羊(♂)与巴什拜羊(♀)级进杂交的方法引进野生盘羊血统。但发现其杂交后代与处在同一饲养条件下的盘羊和巴什拜羊相比,易感染传染性胸膜肺炎而死亡。推测可能是杂交羔羊的某些与疾病抗性相关的基因发生了变异。为此,本研究选择TLR9和ISG15疾病抗性相关基因进行比较基因组学分析,以探索杂交羊抗病力减弱的机理。此外,从DNA水平上研究动物遗传多样性和物种间进化关系已成为分子生物学研究的热点,而盘羊是唯一分布于我国的野生绵羊,但对其遗传多样性及其与我国家养绵羊进化关系的研究还不深入。鉴于此,本研究对野生盘羊mtDNA D-loop区进行了分析,为进一步阐明我国野生盘羊遗传多样性及其与我国家养绵羊的进化关系奠定基础。1、野生盘羊和巴什拜羊及其杂交羊ISG15基因的克隆与序列分析为了探讨疾病抗性相关基因ISG15的mRNA、蛋白保守结构域、3-D结构差异。本研究分三段克隆了这个基因的全长序列,并通过比较基因组学的方法对序列进行了分析。结果显示,新疆野生盘羊、巴什拜羊、F1和F2代ISG15基因总长度分别约为2117bp、2123bp、2074bp、2121bp;新疆野生盘羊ISG15基因mRNA与多物种比对序列的相似性分别为：绵羊93.2%、F1代97.1%、F2代96.9%巴什拜羊97.8%、牛88.8%、猪80.6%、猫79.6%、人67.7%、小鼠66.5%、鱼45.5%、；新疆野生盘羊ISG15基因编码氨基酸与多物种的相似性分别为：绵羊99.2%、F1代99.6%、F2代98.7%、巴什拜羊99.6%、牛95.3%、猪82.3%、猫79.6%、人76%、小鼠73.8%、鱼49.4%；将各物种ISG15蛋白保守结构域进行比较分析发现,在功能域的数量、位置上各物种间都存在差异,其中,盘羊、巴什拜羊的ISG15蛋白存在两个ubiquitin多功能保守域,而F1代羊、F2代羊只含有一个ubiquitin多功能保守域；盘羊、巴什拜羊ISG15蛋白3-D结构的折叠明显少于F1、F2代杂交羊。可能正是由于杂交后代的ISG15基因在mRNA序列,蛋白保守结构域数量,3-D结构上与父母代的ISG15基因出现了差异,而导致了杂交代的ISG15基因在先天免疫和抗病中的作用发生了变化,进而使杂交羔羊抗病力减弱。2、野生盘羊和巴什拜羊及其杂交羊TLR9基因的克隆与序列分析为了探讨疾病抗性相关基因TLR9的mRNA、蛋白保守结构域、3-D结构。本研究分三段克隆了这个基因的全长序列,并通过比较基因组学的方法对序列进行了分析。结果显示,新疆野生盘羊、巴什拜羊、F1和F2代TLR9基因DNA总长度分别为3193bp、3169bp、3103bp和3109bp；新疆野生盘羊TLR9基因mRNA与多物种比对序列的相似性分别为：绵羊99.9%、山羊99%、F1代96.9%、F2代97.1%、巴什拜羊96.9%、牛95.2%、猪85.1%、猫84%、犬83.7%、马83.4%、人79.4%、猴79.1%、小鼠73.1%,新疆野生盘羊TLR9基因编码氨基酸与其他各物种的比对结果分别为：绵羊97.7%、山羊97%、F1代99.6%、F2代98.7%巴什拜羊99.6%、牛94.6%、猪86.2%、猫84%、犬83.7%、马83.4%、人79.4%、猴79.1%、小鼠73.1%；多物种TLR9蛋白功能域分析显示,物种间TLR9蛋白功能域的数量、种类及相对位置上都存在差异,新疆野生盘羊、巴什拜羊与杂交羊之间差异更明显,即新疆野生盘羊、巴什拜羊存在一个LLR RI和一个TLR共计2个功能域,而F1、F2代杂交羊存在一个LLR RI功能域；新疆野生盘羊和巴什拜羊TLR9蛋白的3-D结构是由胞外段富含亮氨酸的重复序列((LRR)和胞内段TIL(Toll/IL IR)结构域构成的,而F1、F2代杂交羊TLR9蛋白的3-D结构在线预测不出来。综上所述,野生盘羊、巴什拜羊与杂交羊TLR9基因的mRNA序列,蛋白保守结构域数量,3-D结构存在差异。因此,推测来自野生盘羊和巴什拜羊的TLR9基因在其杂交后代中的表达与其父、母本间存在差异,从而导致杂交羔羊抗病力减弱,进而使杂交羔羊在幼龄阶段易感染传染性胸膜肺炎。3、野生盘羊mtDNA D-loop区的克隆与序列分析为探讨新疆天山亚种野生盘羊mtDNA D-loop序列多样性及其与我国家养绵羊的亲缘关系。通过PCR扩增的方法特异性扩增了新疆天山亚种野生盘羊线粒体(mtDNA)控制区(D-loop)全长序列片段,PCR产物经凝胶回收纯化后测序并进行序列分析。结果显示,1号和2号盘羊序列全长分别为1070bp和1169bp；在检测的2个个体上共发现73个突变位点,其中有7个插入,50个转换,11个颠换,5个缺失,表明碱基的替换有明显偏倚；1号和2号新疆天山亚种野生盘羊mtDNA D-loop区核苷酸突变位点分别为35个和38个,核苷酸变异率分别为3.27%和3.25%,这一结果表明新疆天山亚种野生盘羊群体线粒体D-loop区存在着丰富的多态性,说明了我国新疆野生盘羊遗传资源比较丰富。通过对野生盘羊和其它绵羊的系统进化树分析发现,野生盘羊与我国家养绵羊的亲缘关系较远,初步推测其并非我国家养绵羊的祖先。更多还原

【Abstract】 Wild Argali is a superordinary wildlife resource in Xinjiang,China.The breed have many features,for example, macro-somatotype、better anti-disease、better accommodation、better toc-flesh and high lean meat rate, and so on. In order to utilize and protect wild animal resource of xinjiang, import blood lineage of Wild Argali largely, the experiment use Wild Argali ((?)),BaShenBai ((?)) or F1 ((?)) for crossing by artificial insemination fecundation The results show that they give birth to crossing sheep of F1、F2,but the anti-disease of F1、F2 is poor.While Wild Argali,BaShenBai have no disease at the same raising condition.So we presume that some disease resistance relational genes of crossing lamb appear variation.Therefore, the TLR9和ISG15 disease resistance relational genes were studied by comparative genomics way,to research the mechanism of the poor anti-disease power of F1、F2 crossing lamb. In addition,It was a hot point which was in molecular biology to research the diversity of animal heredity and the evolution relation among species in the level of DNA,as the research of other animals in this feild,the research in Wild Argali which was belonged to our country had research shallowly.Therefore,we analyzed the Wild Argali mtDNA D-loop region,for the elucidation of the diversity of animal heredity and the evolution relation of Wild Argali in our country.1.Wild Argali、BaShenBai and crossing sheep ISG15 Cloning and Sequence AnalysisThe experiment aims at studying for the mRNA、conservative structural domain、3-D structure of ISG15. The ISG15 complete sequences were specificitly amplified by PCR technology.The PCR product was purified by agarose.The ISG15 complete sequences were sequenced and analysised by the method of comparative genomics. The result showed the length of ISG15 complete sequence were 2117bp、2123bp、2074bp、2121bp,respectively,in wild argali、Bashenbai、F1、F2; The Wild Argali Xinjiang ISG15 mRNA sequence had 94.7% identity with the sheep ISG15 mRNA, and cow88.8%, F1 97.1%, pig 80.6%,cat 76.6%, human 67.7%,mouse 66.5%,fish 45.5%. The Wild Argali Xinjiang ISG15 protein sequence had 99.3% identity with the sheep, and cow 89.1%, Fl 92.8%, pig 74.1%,cat 72.1%, human 63.6%,mouse 63.0%,fish 29.0%. The conserved domain of ISG15 were analysised by the method of comparative genomics. The result showed the quantity, type and the relative position of ISG15 protein motif were different in several species.ISG15 contains two domains with structural homology close to ubiquitin, and species protein 3-D structures were also conservative.Interestly, ISG15 contains one UBQ domain and one ubiquitin multifunction domain in several species, except for F1 which contains one UBQ multifunction domain. The investigation presumed that because of the difference of filial generationISG15gene in the mRNA sequence the quantity of protein conservative stuctural domains and the 3D structure between parental generartion ISG15 gene,resulted the effect of filial generation ISG15gene variancein innate immunity and anti-disease.So the anti-disease of filial generation became weak.2.Wild Argali、BaShenBai and crossing sheep TLR9 Cloning and Sequence AnalysisThe experiment aims at studying for the mRNA、conservative structural domain、3-D structure of TLR9. The TLR9 complete sequences were specificitly amplifited by PCR technology.The PCR product was purified by agarose.The TLR9 complete sequences were sequenced and analysised by the method of comparative genomics. TLR9 were 3193bp、3169bp、3103bp和3109bp.After gene predicted, The Wild Argali Xinjiang TLR9 mRNA sequence had 99.9% identity with the sheepTLR9 mRNA, and goat 99%,cow 95.2%, pig 85.1%, cat 84%, dog 83.7%, horse 83.4%, human79.4%,monkey 79.1%,mouse 73.1%. The Wild Argali Xinjiang TLR9 protein sequence had 97.7% identity with the sheep TLR9 protein, and goat97%,cow 94.6%, pig 86.2%, cat 84%, dog83.7%, horse 83.4%, human79.4%,monkey 79.1%,mouse 73.1%.The conserved domain of TLR9 were analysised by the method of comparative genomics. The result showed the quantity, type and the relative position of TLR9 protein motif were different in several species. The 3-D of TLR9 was constructived by LRR and TIL(Toll/IL IR) structural domain.The above-mentioned structural feature offered theory accordings to research TLR9 of Wild Argali (O.a.karelini),Xinjiang,China.The investigation presumed that the quantity, type,the relative position of TLR9 conservative structural domain and 3-D structure rch of TLR9 protein motif,which came from Wild Argali and BaShenBai, might occurred distinction on crossing lamb.it led to decrease shaply the anti-disease power of crossing lamb,and then it easily infected infectiousness pleuropneumonia in the immature stage of crossing lamb. 3.Wild Argali mitochondria displacement loop region Cloning and Sequence AnalysisThe experiment aims at studying Mitochondrial DNA (mtDNA) Genetic Diversity of Wild Argali.The mtDNA D-loop complete sequences of Wild Argali (O.a.karelini),Xinjiang,China were specificitly amplifited by PCR technology.The PCR product was purified by agarose.The mtDNA D-loop sequences were sequenced and analysised. The result showed the length of mtDNA D-loop complete sequence was 1070bp and 1169bp,respectively,in number one and number two.There were 7 insertion loci,50 transitions loci, lltransversion loci and 5 deletions loci 70 mutational loci which were found in two wild Argalis and transitions was the major mutational type.The mutational loci of mtDNA D-loop sequence, from number one and number two Wild Argali (O.a.karelini), were 35 and 38; mutational ratewere 3.12% and 3.25%.It showed that the Wild Argali (O.a.karelini) population has high variation in mtDNA D-loop sequence.Meanwhile,it further showed that there was plentiful genetic resource of Wild Argali Xinjiang,China. On the basis of the analysis of system evolution tree between Wild Argali and other sheep,the relation between Wild Argali and other sheep was far.In speculation Wild Argali was not the ancestor of our country domesticated sheep.更多还原