基于SNP的捻轉(zhuǎn)血矛線蟲（H.contortus）遺傳多樣性分析及P-糖蛋白基因核苷酸差異位點(diǎn)篩選
發(fā)布時(shí)間：2018-08-08 21:34
【摘要】：捻轉(zhuǎn)血矛線蟲病是畜群中流行較為廣泛的寄生蟲病。該病主要由寄生于牛、羊等反芻動(dòng)物真胃及小腸的捻轉(zhuǎn)血矛線蟲引起。此病在世界范圍內(nèi)均有發(fā)生,常造成放牧動(dòng)物貧血、水腫、衰弱和消化紊亂,對(duì)畜牧業(yè)造成巨大經(jīng)濟(jì)損失。目前,捻轉(zhuǎn)血矛線蟲病的防控仍然以抗蠕蟲類的藥物治療為主要形式,且頻繁用藥有可能使得抗藥性蟲株產(chǎn)生與擴(kuò)散。因此,發(fā)現(xiàn)捻轉(zhuǎn)血矛線蟲的抗藥標(biāo)記分子、探索其抗藥機(jī)制,對(duì)于合理用藥和新藥物開發(fā)具有重要的指導(dǎo)意義。而抗藥性的研究與種群遺傳多樣性有著密切關(guān)系。已有研究顯示,線蟲種群的遺傳多樣性明顯受地理隔離影響,加之我國(guó)放牧動(dòng)物呈現(xiàn)流動(dòng)性加強(qiáng)的趨勢(shì),因此,研究捻轉(zhuǎn)血矛線蟲的種群遺傳多樣性,將大大有利于揭示其種群遺傳差異和指導(dǎo)抗藥性相關(guān)研究。目前對(duì)于捻轉(zhuǎn)血矛線蟲種群遺傳學(xué)和抗藥性標(biāo)記位點(diǎn)的研究,主要是通過單個(gè)特征性標(biāo)記位點(diǎn)實(shí)現(xiàn)的,包括微衛(wèi)星、線粒體基因及核糖體基因等。而這些方法都需要極大的樣本數(shù)目和多次重復(fù)實(shí)驗(yàn)來(lái)完成,且存在一定的穩(wěn)定性誤差。更為重要的是蟲體抗藥性的產(chǎn)生往往涉及多基因多性狀的適應(yīng)和變化,因此,如何同時(shí)發(fā)現(xiàn)蟲體的上述多基因變異特征,成為研究者們關(guān)注的前沿和亟待解決的難題。近年來(lái),隨著下一代測(cè)序技術(shù)(next-generation sequencing)的發(fā)展,為研究者們提供了在基因組范圍內(nèi)探索多基因復(fù)雜性狀的有力工具。因此,本研究首次采用高通量測(cè)序的方法,在全基因組范圍內(nèi)對(duì)捻轉(zhuǎn)血矛線蟲的單核苷酸多態(tài)性(SNP)進(jìn)行評(píng)估,并通過核苷酸差異位點(diǎn)變化來(lái)研究種群的遺傳多樣性及篩選抗藥性相關(guān)基因或標(biāo)記分子,旨在為深入探索捻轉(zhuǎn)血矛線蟲種群基因組學(xué)(population genomics)和發(fā)現(xiàn)抗藥性標(biāo)志分子提供基礎(chǔ)資料。通過基于SNP的方法,選取中國(guó)湖北、內(nèi)蒙、西藏和四川的現(xiàn)場(chǎng)蟲株以及英國(guó)的抗藥蟲株、澳大利亞的標(biāo)準(zhǔn)蟲株為研究對(duì)象,通過第二代測(cè)序技術(shù)2b-RAD對(duì)不同種群蟲株進(jìn)行測(cè)序,獲得不同種群的SNP位點(diǎn),通過比較不同地區(qū)或不同株系捻轉(zhuǎn)血矛線蟲的SNP數(shù)據(jù),分析這些實(shí)驗(yàn)蟲株的種群遺傳多樣性特征,發(fā)現(xiàn)抗伊維菌素蟲株P(guān)-糖蛋白基因的抗藥性相關(guān)位點(diǎn)。結(jié)果表明:種群內(nèi)部存在較低程度的遺傳分化;中國(guó)地區(qū)4個(gè)現(xiàn)場(chǎng)株種群間處于中等遺傳分化程度,中國(guó)地區(qū)4個(gè)現(xiàn)場(chǎng)株種群、澳大利亞標(biāo)準(zhǔn)株種群及英國(guó)抗藥株種群之間處于較大的遺傳分化程度。說(shuō)明不同國(guó)家地理起源的種群,由于受地理隔離的影響,遺傳分化水平較高;而我國(guó)國(guó)內(nèi)的不同地理株種群,由于地理隔離水平受限,遺傳分化水平適中;且西藏種群由于特殊的地理、氣候條件,與其他國(guó)內(nèi)株的遺傳差異水平略高。同時(shí),通過基于對(duì)敏感蟲株種群和抗藥蟲株種群的SNP抗藥標(biāo)記位點(diǎn)的篩選研究,找到了與抗伊維菌素相關(guān)的兩個(gè)標(biāo)記位點(diǎn),一個(gè)為P-糖蛋白基因的SNP標(biāo)記位點(diǎn)(該位點(diǎn)在基因組上的位置為,scaffold HF967529.1,NO.84514,位于P-糖蛋白基因的內(nèi)含子區(qū)域),堿基突變類型為由胞嘧啶(C)突變?yōu)橄汆堰?A);另一個(gè)為與P-糖蛋白基因同一片段上的,位于P-糖蛋白基因附近的SNP位點(diǎn),堿基突變類型為胸腺嘧啶(T)突變?yōu)榘奏?C)。本研究為在全基因組范圍內(nèi)深入探索捻轉(zhuǎn)血矛線蟲群體基本遺傳結(jié)構(gòu)特征及篩選抗藥性標(biāo)志分子等提供了基礎(chǔ)數(shù)據(jù)。
[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.
【學(xué)位授予單位】：上海師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.7
本文編號(hào)：2173102
[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.
【學(xué)位授予單位】：上海師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S852.7
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