基于SNP的捻转血矛线虫（H.contortus）遗传多样性分析及P-糖蛋白基因核苷酸差异位点筛选

发布时间：2018-08-08 21:34

【摘要】：捻转血矛线虫病是畜群中流行较为广泛的寄生虫病。该病主要由寄生于牛、羊等反刍动物真胃及小肠的捻转血矛线虫引起。此病在世界范围内均有发生,常造成放牧动物贫血、水肿、衰弱和消化紊乱,对畜牧业造成巨大经济损失。目前,捻转血矛线虫病的防控仍然以抗蠕虫类的药物治疗为主要形式,且频繁用药有可能使得抗药性虫株产生与扩散。因此,发现捻转血矛线虫的抗药标记分子、探索其抗药机制,对于合理用药和新药物开发具有重要的指导意义。而抗药性的研究与种群遗传多样性有着密切关系。已有研究显示,线虫种群的遗传多样性明显受地理隔离影响,加之我国放牧动物呈现流动性加强的趋势,因此,研究捻转血矛线虫的种群遗传多样性,将大大有利于揭示其种群遗传差异和指导抗药性相关研究。目前对于捻转血矛线虫种群遗传学和抗药性标记位点的研究,主要是通过单个特征性标记位点实现的,包括微卫星、线粒体基因及核糖体基因等。而这些方法都需要极大的样本数目和多次重复实验来完成,且存在一定的稳定性误差。更为重要的是虫体抗药性的产生往往涉及多基因多性状的适应和变化,因此,如何同时发现虫体的上述多基因变异特征,成为研究者们关注的前沿和亟待解决的难题。近年来,随着下一代测序技术(next-generation sequencing)的发展,为研究者们提供了在基因组范围内探索多基因复杂性状的有力工具。因此,本研究首次采用高通量测序的方法,在全基因组范围内对捻转血矛线虫的单核苷酸多态性(SNP)进行评估,并通过核苷酸差异位点变化来研究种群的遗传多样性及筛选抗药性相关基因或标记分子,旨在为深入探索捻转血矛线虫种群基因组学(population genomics)和发现抗药性标志分子提供基础资料。通过基于SNP的方法,选取中国湖北、内蒙、西藏和四川的现场虫株以及英国的抗药虫株、澳大利亚的标准虫株为研究对象,通过第二代测序技术2b-RAD对不同种群虫株进行测序,获得不同种群的SNP位点,通过比较不同地区或不同株系捻转血矛线虫的SNP数据,分析这些实验虫株的种群遗传多样性特征,发现抗伊维菌素虫株P-糖蛋白基因的抗药性相关位点。结果表明:种群内部存在较低程度的遗传分化;中国地区4个现场株种群间处于中等遗传分化程度,中国地区4个现场株种群、澳大利亚标准株种群及英国抗药株种群之间处于较大的遗传分化程度。说明不同国家地理起源的种群,由于受地理隔离的影响,遗传分化水平较高;而我国国内的不同地理株种群,由于地理隔离水平受限,遗传分化水平适中;且西藏种群由于特殊的地理、气候条件,与其他国内株的遗传差异水平略高。同时,通过基于对敏感虫株种群和抗药虫株种群的SNP抗药标记位点的筛选研究,找到了与抗伊维菌素相关的两个标记位点,一个为P-糖蛋白基因的SNP标记位点(该位点在基因组上的位置为,scaffold HF967529.1,NO.84514,位于P-糖蛋白基因的内含子区域),碱基突变类型为由胞嘧啶(C)突变为腺嘌呤(A);另一个为与P-糖蛋白基因同一片段上的,位于P-糖蛋白基因附近的SNP位点,碱基突变类型为胸腺嘧啶(T)突变为胞嘧啶(C)。本研究为在全基因组范围内深入探索捻转血矛线虫群体基本遗传结构特征及筛选抗药性标志分子等提供了基础数据。
[Abstract]:Spear disease is a widespread parasitic disease in the herd. The disease is mainly caused by parasitic on the real stomach and small intestine of ruminant animals, such as cattle, sheep and other ruminants. This disease occurs all over the world. It is often made into animal animal anemia, dropsy, weak and digestive disorder, causing huge economic loss to animal husbandry. At present, twisting The prevention and control of spear nematode disease is still in the main form of antiworm drug treatment, and frequent use of drugs may lead to the production and diffusion of drug-resistant strains. Therefore, the discovery of anti drug marker molecules of spear spear nematode and the exploration of its anti drug mechanism have important guiding significance for rational drug use and the development of new drugs. Studies have been closely related to the genetic diversity of the population. Studies have shown that the genetic diversity of the nematode population is obviously affected by geographical isolation, and the grazing animals in our country show a trend of enhanced mobility. Therefore, the study of the genetic diversity of the population of spear spear nematodes will be greatly beneficial to reveal the genetic diversity of the population and to guide the resistance to drug resistance. Research on the population genetics and resistance marker loci of the spear nematode, mainly through single characteristic marker loci, including microsatellites, mitochondrial genes and ribosome genes. These methods require a large number of samples and repeated experiments to achieve a certain stability. It is more important that the production of insect resistance is often involved in the adaptation and change of multi gene and multiple traits. Therefore, how to find the characteristics of the multigene mutation of the insect body at the same time has become the frontier of attention and a difficult problem to be solved. In recent years, with the development of the next generation sequencing technology (next-generation sequencing), Researchers have provided a powerful tool for exploring multi gene complex traits within the genome. Therefore, this study is the first to use high throughput sequencing to evaluate the single nucleotide polymorphisms (SNP) of spear spear nematode in the whole genome, and to study the genetic diversity of the population by the variation of nucleotide diversity sites. Screening resistance related genes or marker molecules to provide basic information for the in-depth exploration of the population genomics and drug resistant marker molecules. Through the SNP based method, the present field insects in Hubei, Inner Mongolia, Tibet and Sichuan, China, and the British antidrug strains, and the Australian standard are selected. The second generation sequencing technology, 2b-RAD, was used to sequence the different populations of the population and to obtain the SNP loci of different populations. By comparing the SNP data of different regions or different strains of spear spear nematode, the genetic diversity of these strains was analyzed, and the resistance to the P- glycoprotein gene of the anti ivermectin strain was found. The results showed that there was a low degree of genetic differentiation within the population; 4 locale populations in China were in the middle genetic differentiation, 4 in China, and between the Australian standard plant population and the British antidrug-resistant strain population, which indicated the geographical origin of different countries. Population, the genetic differentiation level is high due to geographical isolation, and the genetic differentiation level of different geographical strains in China is moderate due to the limited geographic isolation level, and the Tibet population is slightly higher in genetic difference with other domestic plants due to special geographical and climatic conditions. The screening of the anti drug marker loci of the anti drug strain SNP found two marker loci related to the anti ivermectin, a SNP marker for the P- glycoprotein gene (the location of the site in the genome, scaffold HF967529.1, NO.84514, in the introns of the P- glycoprotein gene), and the base mutation type from the cytosimon. The C mutation is adenine (A); the other is the SNP site near the P- glycoprotein gene in the same fragment as the P- glycoprotein gene, and the base mutation type is thymine (T) mutation to cytosine (C). This study is to explore the basic genetic structure characteristics of the group of spear spear nematode and the screening of drug resistance markers in the whole genome area. The molecules and so on provide basic data.
【学位授予单位】：上海师范大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.7

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