白纹伊蚊COI基因多态性分析及AGO2和Dcr-2基因克

发布时间：2018-04-27 05:00

  本文选题：白纹伊蚊 + 细胞色素C氧化酶亚基Ⅰ基因　； 参考：《南方医科大学》2012年硕士论文

【摘要】：蚊虫(Mosquito)是自然界中一类最常见的昆虫,在分类上隶属于昆虫纲(Class Insecta Linnaeus),双翅目(Diptera),蚊科(Culicidae),是多种重要疾病的传播媒介。蚊分为三个亚科(subfamilies):巨蚊亚科(Toxorhyncitinae)、按蚊亚科(Anophelinae)和库蚊亚科(Culicinae),其中按蚊亚科和库蚊亚科的许多蚊虫能传播病原体,是医学上重要媒介昆虫或称为病媒昆虫。 蚊虫不仅吸血滋扰人类,影响人们的正常工作与休息,还传播多种蚊媒病,直接威胁人类的健康与生命,在世界上大部分地区已成为严重的公共卫生问题。以蚊虫作为传播媒介的病原体要顺利从一个动物宿主传播至另一动物宿主,必须在蚊体内建立有效的繁殖传播模式并同时避免杀死传播媒介。蚊虫传播病原体的能力受到其遗传因素的控制。在长期的进化过程中蚊虫不断受到环境的选择压力,导致种群间基因交流、种群遗传结构出现变化,产生遗传多样性,出现一个种群内也可存在两种以上不同遗传类型的个体。了解它们的差异与传播疾病的能力之间的关系,对蚊媒病的防控具有重要的意义。 利用多种分子生物学技术对蚊虫的种群进行遗传分析,调查不同地区蚊虫种群遗传结构和基因流程度,对于控制虫媒传染病具有重要的价值。Kamgang等对非洲喀麦隆南部12个采样地的白纹伊蚊6个微卫星位点和2个线粒体基因ND5和COI遗传多样性进行分析,发现喀麦隆白纹伊蚊种群低水平的遗传变异与近年该物种多次入侵及扩散到该国的现象相关联,其自然种群的遗传结构指向白纹伊蚊由热带地区引入。线粒体DNA在昆虫等生物体内广泛存在,可作为遗传差异分析和特定病毒传播媒介识别的有效标记。 蚊虫对病原体的感染能力固然受到自身遗传因素的影响,蚊虫感染病原生物的过程中,实际上也是病原生物侵袭与蚊自身防御的相互斗争过程。RNA干扰作为蚊虫自然抗病毒免疫效应,在生物抗病毒感染、生物发育时序的控制、细胞生长和凋亡等过程中发挥着重要的作用。由siRNA介导的RNA干扰抗病毒效应已在多种媒介昆虫中发现,其中AG02和Dcr-2基因起着关键作用,通过控制或改造媒介RNAi抗病毒效应成为分析媒介和病毒间相互作用的常用手段。如Vargas等通过RNAi沉默AG02、Dcr-2和R2D2基因的表达后,能增加登革2型病毒(Dengue virus type2, DENV2)在埃及伊蚊体内的复制同时缩短病毒传播的外潜伏期。 本文尝试对广州市白纹伊蚊COI基因遗传多态性进行分析,从分子水平上探索其与登革热流行的关系；并尝试对白纹伊蚊RNAi通路中重要的两个关键基因AG02和Dcr-2用简并引物进行体外克隆,初步分析它们在白纹伊蚊不同发育阶段和感染登革前后的转录水平,以了解RNAi在白纹伊蚊抗病毒效应中的作用机制,对评估白纹伊蚊的媒介易感效应有着重要的参考价值。 目的： 1.分析广州市内不同群体白纹伊蚊细胞色素C氧化酶亚基I基因遗传多态性。 2.克隆白纹伊蚊AG02和Dcr-2基因片段并进行生物信息学分析。 3.分析各发育阶段白纹伊蚊AG02和Dcr-2基因的转录水平。 4.白纹伊蚊雌蚊经胸腔注射感染登革2型病毒,分析感染前后AG02和Dcr-2基因的转录水平差异。 方法： 1.2010年9月采集并鉴定广州市12个点的白纹伊蚊成蚊和幼虫。 2.单蚊提取白纹伊蚊雌蚊基因组,扩增细胞色素C氧化酶亚基I基因并纯化PCR产物。 3.所有样本COI基因PCR产物经TaqI酶切成小片段,用单链构象多态性方法筛选电泳条带有差异的样本。 4.筛选的样品连接pMD18-T载体、克隆测序,获得序列样本进行变异位点分析,并构建COI基因系统进化树。 5.利用CODEHOP方法设计AG02和Dcr-2基因的简并引物,以白纹伊蚊雌蚊cDNA为模板克隆目的基因片段,并对测序结果进行生物信息学分析。 6.设计扩增AG02和Dcr-2基因特异性引物,分析不同发育阶段白纹伊蚊AG02和Dcr-2基因的转录水平。 7.乳鼠脑内接种登革2型病毒传代,研磨收集感染病毒乳鼠脑组织上清。 8.用乳鼠病毒上清液感染C6/36细胞繁殖登革2型病毒,并测定病毒滴度。 9.建立经胸腔注射病毒到白纹伊蚊体内的人工感染方法,制备一批感染登革2型病毒的雌性白纹伊蚊。 10.分别在白纹伊蚊羽化后2天(设为对照组)和感染登革2型病毒后第1、3、5、7、9、11、14天提取总RNA,用半定量RT-PCR的方法检测AG02和Dcr-2基因的相对转录水平,用ImageJ图像分析软件计算条带灰度值,收集数据并进行统计学分析。 结果： 1.成功克隆不同白纹伊蚊个体COI基因并被TaqI酶切成3个小片段。 2.利用单链构象多态性对采集的白纹伊蚊样本进行筛选,选出13个电泳条带有差异的个体。 3.13条序列克隆测序,所得序列长度均为709bp。A+T碱基的平均含量为67.42%,符合昆虫线粒体A、T碱基偏好性的基因规律。COI基因序列中保守位点697个,变异位点12个(1.69%),其中9处出现转换,3处出现颠换。COI序列间遗传距离的范围在0.000-0.007之间。 4.构建的COI基因进化树显示,广州市不同采样点白纹伊蚊线粒体COI基因存在差异,从化、番禺、南沙和增城采样点的白纹伊蚊与天河单独聚成一类,萝岗区与白云区采样点的蚊株聚成一类；白纹伊蚊不同种群的所有个体稳定的形成高支持率的单系群。 5.简并引物扩增白纹伊蚊AG02和Dcr-2基因片段长度分别为326bp和491bp,同源性及生物信息学分析确定为目的基因。 6.AGO2和Dcr-2基因在白纹伊蚊卵、Ⅰ/Ⅱ龄幼虫、Ⅲ/Ⅳ龄幼虫、蛹、雄蚊和雌蚊等时期均有转录,且雌蚊的转录水平均高于其他时期。 7.成功在乳鼠和C6/36细胞中繁殖登革2型病毒,测得未稀释的病毒滴度为10671TCID50/0.1ml。 8.白纹伊蚊感染病毒前和感染后1-14天内AG02基因转录水平无明显差异,感染后第7天和第14天Dcr-2基因转录水平显著升高(P0.05与感染前对比),第11天则出现显著降低(P0.05)。 结论： 1.广州市不同群体白纹伊蚊间既有基因交流,也出现部分个体的遗传变异。 2.成功克隆出白纹伊蚊RNA干扰通道关键基因AGO2和Dcr-2的片段,并被Genbank收录,登录号分别为：JQ764670和JQ764671。 3.白纹伊蚊的生长发育过程均出现AG02和Dcr-2基因的参与,显示RNA干扰在蚊生长发育中发挥着重要作用。 4.Dcr-2基因在不同感染时间的转录水平差异,提示病毒在蚊体内感染传播过程受到RNAi的抗病毒效应的影响。
[Abstract]:Mosquito is one of the most common types of insects in nature, belonging to the Class Insecta Linnaeus (Diptera) and mosquito (Culicidae). It is a medium for many important diseases. The mosquito is divided into three subfamilies (subfamilies): Mega mosquito (Toxorhyncitinae), Anopheles subfamily (Anophelinae) and Cu Yako (Culicina). E). Many mosquitoes of Anopheles subfamily and Culex subfamily can transmit pathogens and are important medical vectors or vectors.
Mosquitoes not only suck blood to human, affect people's normal work and rest, but also spread a variety of mosquito borne diseases, which directly threaten the health and life of human beings. In most parts of the world, it has become a serious public health problem. The pathogen of mosquito as a medium of transmission should be transmitted from one animal host to another. To establish effective propagation mode in the body of mosquitoes and avoid killing the media at the same time, the ability of mosquitoes to spread pathogens is controlled by their genetic factors. In the long period of evolution, mosquitoes are constantly subjected to environmental pressure, resulting in genetic communication among populations, changes in population genetic structure, genetic diversity, and emergence of a genetic diversity. There are more than two individuals with different genetic types in the population. It is of great significance to understand the relationship between their differences and the ability to spread the disease, and to prevent and control mosquito borne diseases.
A variety of molecular biology techniques are used to analyze the population of mosquitoes and investigate the genetic structure and gene flow of mosquitoes in different regions. It is of great value to control the insect vector infectious diseases, such as.Kamgang and 6 microsatellite loci of Aedes albopictus and 2 mitochondrial genes, ND5 and COI, in the southern part of Cameroon, Africa. Diversity analysis showed that the low level genetic variation of Aedes albopictus population in Cameroon was associated with the multiple invasion and diffusion of the species to the country in recent years. The genetic structure of the natural population was introduced into the tropical area. The mitochondrial DNA is widely existed in the insect and other organisms, which can be used as a genetic difference analysis and special analysis. An effective marker for the identification of viral vectors.
The infection ability of mosquitoes to pathogens is influenced by their own genetic factors. In the process of mosquito infection, in fact, it is also the mutual struggle between the pathogen invasion and the mosquito's own defense..RNA interference as the natural antiviral immunity effect of mosquitoes, the control of the biological resistance to disease, the control of the biological development time sequence and the cell growth. The antiviral effect of RNA interference mediated by siRNA has been found in a variety of media insects, and AG02 and Dcr-2 genes play a key role. By controlling or modifying the RNAi antiviral effect of media RNAi, it is a common means to analyze the interaction between vectors and viruses. For example, Vargas and so on through RNAi silencing AG0 2, the expression of Dcr-2 and R2D2 genes can increase the replication of the dengue type 2 virus (Dengue virus type2, DENV2) in Aedes aegypti and shorten the external incubation period of the virus transmission.
This paper tries to analyze the genetic polymorphisms of the COI gene of Aedes albopictus in Guangzhou and explore its relationship with dengue fever at the molecular level, and try to clone the two important genes of Aedes albopictus RNAi pathway, AG02 and Dcr-2, by degenerate primers, and preliminarily analyze their different developmental stages and sense in Aedes albopictus. The transcriptional level before and after dyed dengue is used to understand the mechanism of RNAi in the antiviral effect of Aedes albopictus, which is of great reference value for evaluating the vector susceptibility of Aedes albopictus.
Objective:
1. to analyze the genetic polymorphism of I gene of cytochrome C oxidase subunit of Aedes albopictus from different populations in Guangzhou.
2. cloning of AG02 and Dcr-2 fragments of Aedes albopictus and bioinformatics analysis.
3. to analyze the transcriptional levels of AG02 and Dcr-2 genes in Aedes albopictus at different developmental stages.
4. dengue virus 2 was injected into the thoracic cavity of Aedes albopictus. The transcriptional levels of AG02 and Dcr-2 genes were analyzed before and after infection.
Method锛，

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