巴基斯坦绵羊和山羊朊蛋白基因多态性分析

发布时间：2018-02-26 19:35

本文关键词： 传染性海绵状脑病朊蛋白绵羊山羊系谱树多态性分析　出处：《中国农业大学》2016年博士论文　论文类型：学位论文

【摘要】：传染性海绵状脑病(Transmissible Spongiform Encephalopathie, TSEs)是一组可引起神经退行性变性的致命疾病,包括鹿和麇鹿的慢性消耗性疾病(Chronic Wasting Disease, CWD),人的克雅氏病(Creutzfeldt-Jakob disease, CJD),水貂中的传染性水貂脑病(TME),牛的疯牛病和绵羊及山羊的羊痒病等。异常形式的PrPC的蓄积是引起TSE疾病的主要原因。这种异常形式的PrP被称为PrPSc,其具有部分蛋白酶抗性和传染性。因此,正常朊蛋白一级结构的改变对于构象异常朊蛋白的产生会有影响。为了阐明巴基斯坦绵羊和山羊不同品种朊蛋白一级结构的多态性,本研究对12个品种的绵羊和11个品种的山羊朊蛋白结构进行测序分析,从而为绵羊和山羊的抗病育种提供理论基础,也为痒病的发生提供详尽的基础数据。首先,本研究从巴基斯坦所有省份选取的涉及十二个品种129只绵羊,进行朊病毒蛋白的基因序列分析,并比较其与他哺乳动物物种的基因多态性。首先,从羊颈静脉无菌采取正常血液样本。采用标准流程从血液样本中提取基因组DNA,然后,对基因组DNA进行了771 bp prion基因片段的PCR扩增。对PCR产物进行测序。通过软件Codon Code aligner来进行序列分析,并检测识别新的单核苷酸多态性。通过软件Codon Code aligner和MEGAv.6来对PrP基因序列进行编辑和匹配,进而构建系谱树,并探查巴基斯坦山羊种群与其他哺乳动物群的系统演化。使用“R’统计软件包用于绘制多维标度图,测算遗传距离并进行主成分分析。单核苷酸多态性分析结果表明在379,380,404,407,414,428,437,455,461,511,512,513,566,572,691,711和718位点发生突变。这些突变表现为转移突变和颠换突变。序列分析表明,绵羊PrP基因出现7个氨基酸多态性位点。这些氨基酸多态性将这些品种绵羊的基因型划分为13个等位基因和15个基因型。羊痒病最耐受基因突变(Q171R)出现的频度为0.107。羊痒病最敏感基因突变(A136 V)在所检测羊样品里没有出现。在接下来的研究中,从巴基斯坦所有省份的11种山羊品种中选取了94只山羊,对其进行朊蛋白基因的检测。通过对PrP基因中771 bp编码区域的PCR扩增,观察了氨基酸亚基的六个多肽位点(一个新的)：126(A-G)、304(G-T)、379 (A-G)、414(C-T)、428 (A-G)和718(C-T)。位点C.428与其它10个品种相比较,具有高度的突变性。比特拉羊(Beetal)与其他10个品种相比,在此位点具有更多的变异性。基于这些PrP变体,NJ系谱树显示鹿与所有山羊种群,连同家绵羊和摩弗伦羊共处一个分支并共享其最近共同祖先(most recent common ancestors, MRCA)。蛋白分析表明这些多态性可以导致不同的一级、二级和三级蛋白结构。多维标度图清晰地表明山羊种群与其他哺乳动物相比较,有较远的生物学相近性并且明显已经分离为特殊的种群。通过主分量分析,绵羊和山羊中朊蛋白基因的这些新发现,将为对羊痒病的抗性和易感性、饲养管理、跨物种传播、发病机制的进一步研究提供帮助。综上所述,本研究对巴基斯坦所有省份的绵羊和山羊进行了调研,涉及12个品种的绵羊和11个品种的山羊,采集了大量的样本,基本反映了巴基斯坦地区山羊及绵羊的基因分布情况,为相关部门防控动物疫病提供重要的依据,尤其是对防控朊病毒在这些动物中的传播具有重要的参考价值和指导意义。这些在山羊和绵羊中朊蛋白基因多态性的发现,为巴基斯坦加强家山羊和家绵羊对痒病的防控提供分子理论依据。
[Abstract]:Transmissible spongiform encephalopathy (Transmissible Spongiform, Encephalopathie, TSEs) is a group of diseases can cause fatal neurodegenerative diseases, including chronic consumption of deer and deer (Chronic Wasting Disease, the CWD), human Creutzfeldt Jakob disease (Creutzfeldt-Jakob disease, CJD), mink in transmissible mink encephalopathy (TME), bovine the BSE and scrapie in sheep and goats. The accumulation of abnormal forms of PrPC is the main cause of TSE disease. The abnormal form of PrP called PrPSc, which is part of the protease resistant and infectious. Therefore, the primary structure of normal prion protein to produce abnormal prion protein conformational change. There will be affected. In order to elucidate the polymorphism of Pakistan sheep and goat prion protein of different varieties of the primary structure of goat prion protein in this study, 12 sheep and 11 varieties of structure sequence analysis, In order to provide a theoretical basis for the breeding of sheep and goat, also provide the basic data for the detailed occurrence of scrapie. Firstly, this study selected from Pakistan province to all twelve varieties of 129 sheep prion protein gene sequence analysis, and compare it with his mammalian species polymorphism firstly. And take the normal blood samples from sheep jugular vein. The standard aseptic process of extracting genomic DNA from blood samples and genomic DNA of the 771 BP prion gene fragment was amplified by PCR. The PCR products were sequenced. Sequence analysis was performed by software Codon Code aligner, and the detection and identification of new single nucleotide polymorphism through. The software Codon Code aligner and MEGAv.6 of PrP gene sequences were edited and matched, and construct phylogenetic tree, and exploration of Pakistan goat population and other mammalian animal group The use of "R" system evolution. The statistical package used to draw a multidimensional scaling map, principal component analysis and calculation of genetic distance. Single nucleotide polymorphism analysis showed that occurred in 379380404407414428437455461511512513566572691711 and 718 mutations. These mutations showed metastasis mutation and transversion mutation. Sequence analysis showed that 7 amino acids of sheep PrP gene polymorphism site. These amino acid polymorphism divides the genotypes of these sheep breeds was 13 alleles and 15 genotypes. The scrapie resistance gene (Q171R) mutation frequency of 0.107. for scrapie sensitive gene mutation (A136 V) in the detection of samples in sheep did not appear. The following research, from 11 goat breeds in Pakistan province were selected in all 94 goats, detection of prion protein gene by PrP based on it. Because of amplification of 771 BP encoding region of PCR, a six amino acid polypeptide sites to observe subunit (a new):126 (A-G), 304 (G-T), 379 (A-G), 414 (C-T), 428 (A-G) and 718 (C-T). The sites of C.428 compared with other 10 varieties phase has mutagenicity. High bit pull the sheep (Beetal) compared with the other 10 varieties, with more variability at this locus. The PrP variant based on NJ family tree of deer and goat populations show all, together with domestic sheep and mouflon coexistence of a branch and share the most recent common ancestor (most recent common ancestors, MRCA). Protein analysis showed that these polymorphisms can lead to different level, two level and three level of protein structure. Multidimensional scaling map clearly and other mammals Mingshan sheep population compared with biological far similar and has clearly separated into special populations. Through principal component analysis, sheep And these new findings of prion protein gene in goats, for feeding and management of scrapie resistance and susceptibility, and cross species transmission, to provide help for further research in pathogenesis. In summary, the study of sheep and goats in Pakistan province all conducted an investigation, involving 12 species and 11 varieties of sheep the goat, collecting a large number of samples, basically reflects the distribution of goats and sheep in Pakistan gene, provide an important basis for the relevant departments to prevention and control of animal diseases, especially has important reference value and guiding significance for the prevention and control of the spread of prions in these animal. These in goats and sheep in the prion protein gene polymorphism the discovery of the goat and sheep home, provide molecular basis on the prevention and control of scrapie for Pakistan to strengthen.

【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S826;S827

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