本文選題：白紋伊蚊 + 細胞色素C氧化酶亞基Ⅰ基因��； 參考：《南方醫(yī)科大學》2012年碩士論文

【摘要】：蚊蟲(Mosquito)是自然界中一類最常見的昆蟲,在分類上隸屬于昆蟲綱(Class Insecta Linnaeus),雙翅目(Diptera),蚊科(Culicidae),是多種重要疾病的傳播媒介。蚊分為三個亞科(subfamilies):巨蚊亞科(Toxorhyncitinae)、按蚊亞科(Anophelinae)和庫蚊亞科(Culicinae),其中按蚊亞科和庫蚊亞科的許多蚊蟲能傳播病原體,是醫(yī)學上重要媒介昆蟲或稱為病媒昆蟲。 蚊蟲不僅吸血滋擾人類,影響人們的正常工作與休息,還傳播多種蚊媒病,直接威脅人類的健康與生命,在世界上大部分地區(qū)已成為嚴重的公共衛(wèi)生問題。以蚊蟲作為傳播媒介的病原體要順利從一個動物宿主傳播至另一動物宿主,必須在蚊體內建立有效的繁殖傳播模式并同時避免殺死傳播媒介。蚊蟲傳播病原體的能力受到其遺傳因素的控制。在長期的進化過程中蚊蟲不斷受到環(huán)境的選擇壓力,導致種群間基因交流、種群遺傳結構出現變化,產生遺傳多樣性,出現一個種群內也可存在兩種以上不同遺傳類型的個體。了解它們的差異與傳播疾病的能力之間的關系,對蚊媒病的防控具有重要的意義。 利用多種分子生物學技術對蚊蟲的種群進行遺傳分析,調查不同地區(qū)蚊蟲種群遺傳結構和基因流程度,對于控制蟲媒傳染病具有重要的價值。Kamgang等對非洲喀麥隆南部12個采樣地的白紋伊蚊6個微衛(wèi)星位點和2個線粒體基因ND5和COI遺傳多樣性進行分析,發(fā)現喀麥隆白紋伊蚊種群低水平的遺傳變異與近年該物種多次入侵及擴散到該國的現象相關聯,其自然種群的遺傳結構指向白紋伊蚊由熱帶地區(qū)引入。線粒體DNA在昆蟲等生物體內廣泛存在,可作為遺傳差異分析和特定病毒傳播媒介識別的有效標記。 蚊蟲對病原體的感染能力固然受到自身遺傳因素的影響,蚊蟲感染病原生物的過程中,實際上也是病原生物侵襲與蚊自身防御的相互斗爭過程。RNA干擾作為蚊蟲自然抗病毒免疫效應,在生物抗病毒感染、生物發(fā)育時序的控制、細胞生長和凋亡等過程中發(fā)揮著重要的作用。由siRNA介導的RNA干擾抗病毒效應已在多種媒介昆蟲中發(fā)現,其中AG02和Dcr-2基因起著關鍵作用,通過控制或改造媒介RNAi抗病毒效應成為分析媒介和病毒間相互作用的常用手段。如Vargas等通過RNAi沉默AG02、Dcr-2和R2D2基因的表達后,能增加登革2型病毒(Dengue virus type2, DENV2)在埃及伊蚊體內的復制同時縮短病毒傳播的外潛伏期。 本文嘗試對廣州市白紋伊蚊COI基因遺傳多態(tài)性進行分析,從分子水平上探索其與登革熱流行的關系；并嘗試對白紋伊蚊RNAi通路中重要的兩個關鍵基因AG02和Dcr-2用簡并引物進行體外克隆,初步分析它們在白紋伊蚊不同發(fā)育階段和感染登革前后的轉錄水平,以了解RNAi在白紋伊蚊抗病毒效應中的作用機制,對評估白紋伊蚊的媒介易感效應有著重要的參考價值。 目的： 1.分析廣州市內不同群體白紋伊蚊細胞色素C氧化酶亞基I基因遺傳多態(tài)性。 2.克隆白紋伊蚊AG02和Dcr-2基因片段并進行生物信息學分析。 3.分析各發(fā)育階段白紋伊蚊AG02和Dcr-2基因的轉錄水平。 4.白紋伊蚊雌蚊經胸腔注射感染登革2型病毒,分析感染前后AG02和Dcr-2基因的轉錄水平差異。 方法： 1.2010年9月采集并鑒定廣州市12個點的白紋伊蚊成蚊和幼蟲。 2.單蚊提取白紋伊蚊雌蚊基因組,擴增細胞色素C氧化酶亞基I基因并純化PCR產物。 3.所有樣本COI基因PCR產物經TaqI酶切成小片段,用單鏈構象多態(tài)性方法篩選電泳條帶有差異的樣本。 4.篩選的樣品連接pMD18-T載體、克隆測序,獲得序列樣本進行變異位點分析,并構建COI基因系統(tǒng)進化樹。 5.利用CODEHOP方法設計AG02和Dcr-2基因的簡并引物,以白紋伊蚊雌蚊cDNA為模板克隆目的基因片段,并對測序結果進行生物信息學分析。 6.設計擴增AG02和Dcr-2基因特異性引物,分析不同發(fā)育階段白紋伊蚊AG02和Dcr-2基因的轉錄水平。 7.乳鼠腦內接種登革2型病毒傳代,研磨收集感染病毒乳鼠腦組織上清。 8.用乳鼠病毒上清液感染C6/36細胞繁殖登革2型病毒,并測定病毒滴度。 9.建立經胸腔注射病毒到白紋伊蚊體內的人工感染方法,制備一批感染登革2型病毒的雌性白紋伊蚊。 10.分別在白紋伊蚊羽化后2天(設為對照組)和感染登革2型病毒后第1、3、5、7、9、11、14天提取總RNA,用半定量RT-PCR的方法檢測AG02和Dcr-2基因的相對轉錄水平,用ImageJ圖像分析軟件計算條帶灰度值,收集數據并進行統(tǒng)計學分析。 結果： 1.成功克隆不同白紋伊蚊個體COI基因并被TaqI酶切成3個小片段。 2.利用單鏈構象多態(tài)性對采集的白紋伊蚊樣本進行篩選,選出13個電泳條帶有差異的個體。 3.13條序列克隆測序,所得序列長度均為709bp。A+T堿基的平均含量為67.42%,符合昆蟲線粒體A、T堿基偏好性的基因規(guī)律。COI基因序列中保守位點697個,變異位點12個(1.69%),其中9處出現轉換,3處出現顛換。COI序列間遺傳距離的范圍在0.000-0.007之間。 4.構建的COI基因進化樹顯示,廣州市不同采樣點白紋伊蚊線粒體COI基因存在差異,從化、番禺、南沙和增城采樣點的白紋伊蚊與天河單獨聚成一類,蘿崗區(qū)與白云區(qū)采樣點的蚊株聚成一類；白紋伊蚊不同種群的所有個體穩(wěn)定的形成高支持率的單系群。 5.簡并引物擴增白紋伊蚊AG02和Dcr-2基因片段長度分別為326bp和491bp,同源性及生物信息學分析確定為目的基因。 6.AGO2和Dcr-2基因在白紋伊蚊卵、Ⅰ/Ⅱ齡幼蟲、Ⅲ/Ⅳ齡幼蟲、蛹、雄蚊和雌蚊等時期均有轉錄,且雌蚊的轉錄水平均高于其他時期。 7.成功在乳鼠和C6/36細胞中繁殖登革2型病毒,測得未稀釋的病毒滴度為10671TCID50/0.1ml。 8.白紋伊蚊感染病毒前和感染后1-14天內AG02基因轉錄水平無明顯差異,感染后第7天和第14天Dcr-2基因轉錄水平顯著升高(P0.05與感染前對比),第11天則出現顯著降低(P0.05)。 結論： 1.廣州市不同群體白紋伊蚊間既有基因交流,也出現部分個體的遺傳變異。 2.成功克隆出白紋伊蚊RNA干擾通道關鍵基因AGO2和Dcr-2的片段,并被Genbank收錄,登錄號分別為：JQ764670和JQ764671。 3.白紋伊蚊的生長發(fā)育過程均出現AG02和Dcr-2基因的參與,顯示RNA干擾在蚊生長發(fā)育中發(fā)揮著重要作用。 4.Dcr-2基因在不同感染時間的轉錄水平差異,提示病毒在蚊體內感染傳播過程受到RNAi的抗病毒效應的影響。
[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錛，

本文編號：1809272