【摘要】：蜱是常見的人和動物體表寄生蟲,作為嗜血性節(jié)肢動物,蜱在分類學上屬于節(jié)肢動物門、蛛形綱、蜱螨目、蜱總科,其生活史復雜,繁殖率高,經(jīng)歷卵、幼蟲、若蟲、成蟲四個發(fā)育階段,屬于不完全變態(tài)發(fā)育。作為多種人獸共患病病原的儲存宿主和傳播媒介,蜱可傳播和感染病原引發(fā)原蟲病、森林腦炎、新疆出血熱、回歸熱、萊姆病、Q熱、立克次體病等。威脅人類健康,對畜牧經(jīng)濟發(fā)展帶來巨大危害。因此,預防和控制蜱的傳播十分重要。鐮形扇頭蜱屬于硬蜱的一種,廣泛分布于我國長江以南,是我國南方的優(yōu)勢蜱種。目前對殺蜱除蜱的主要方法是化學藥物殺滅,這種方法對蜱的殺傷效果雖好,但環(huán)境污染嚴重,毒性藥物對宿主危害性大,隨著生物技術研究的深入,新型環(huán)保的預防控制蜱的策略不斷發(fā)展,如抗蜱疫苗研究等,但還未取得成功,新的防治技術需求還十分迫切。RNA干擾技術作為新興的研究技術,在蜱研究中得到廣泛應用。本實驗室已克隆鑒定了鐮形扇頭蜱抗凝血分子Rhipilin-1,并發(fā)現(xiàn)通過顯微注射證明RNA干擾該基因可有效降低蜱的吸血能力。本研究根據(jù)RNA干擾原理,將Rhipilin-1dsRNA直接浸泡蜱體,通過脂質體浸染傳遞途徑達到RNA干擾效果,從而實施如殺蟲劑滅蜱的新型防治技術:其一,以重組質粒pGEM-T-Rhipilin-1和pGEM-T-Luciferase為模板,根據(jù)T7試劑盒的操作說明,在蜱體外合成目的基因Rhipilin-1dsRNA和對照基因Luciferase dsRNA;其二,在浸泡過程中篩選蜱各階段最佳脂質劑、浸泡時間以及dsRNA與脂質劑浸泡比例并通過熒光標記dsRNA的浸泡觀察dsRNA分布;其三,以最佳脂質劑、浸泡時間、浸泡比例對鐮形扇頭蜱幼蜱、若蜱和成蜱三個階段浸泡,以Luciferase基因的dsRNA作為對照,進行動物體實驗觀察。結果顯示,最佳浸泡條件篩選為幼蜱脂質劑Lipofectamine 2000、浸泡24h、dsRNA與脂質劑體積比為2:1;若蜱脂質劑DMRIE-C、浸泡12h、dsRNA與脂質劑體積比為1:1;幼蜱脂質劑DMRIE-C、浸泡12h、dsRNA與脂質劑體積比為1:2;熒光dsRNA進入蜱體;浸泡后的鐮形扇頭蜱飽血時間顯著增長,飽血體重顯著減少,即對鐮形扇頭蜱原有生物學功能產(chǎn)生顯著影響。因此,可將Rhipilin-1基因作為殺蜱劑的候選基因,這為RNA干擾方法學在蜱防控實踐應用以及新型環(huán)保的殺蜱劑的創(chuàng)制提供基礎。
[Abstract]:Ticks are common human and animal body surface parasites. As haemophilic arthropods, ticks belong to Arthropoda, Arachnidae, Acaridae, family Acari, their life history is complex, reproduction rate is high, they experience eggs, larvae, nymphs. The four developmental stages of adults belong to incomplete metamorphosis. As a storage host and vector of zoonotic pathogens, ticks can transmit and infect pathogens to cause protozoonosis, forest encephalitis, Xinjiang haemorrhagic fever, recurrent fever, Lyme disease, Q fever, Rickettsia disease and so on. It threatens human health and brings great harm to the development of animal husbandry economy. Therefore, it is important to prevent and control the spread of ticks. Acari falciformis is one of the most dominant ticks in southern China, which is widely distributed in the south of the Yangtze River. At present, the main way to kill ticks is to kill the ticks by chemical drugs. Although this method has a good killing effect on ticks, but the environment is polluted seriously and toxic drugs are harmful to the host, with the development of biotechnology, New environmental protection strategies for ticks prevention and control, such as the study of anti-tick vaccine, have been continuously developed, but the need for new control techniques is urgent. As a new research technology, RNA interference technology has been widely used in ticks research. The anticoagulant molecule Rhipilin-1, of ticks falciform fan head has been cloned and identified in our laboratory. Microinjection has proved that RNA interference with this gene can effectively reduce the ability of blood absorption of ticks. According to the principle of RNA interference, Rhipilin-1dsRNA was directly soaked in ticks, and the effect of RNA interference was achieved through liposome immersion and transmission pathway, so as to implement a new control technology such as insecticide to kill ticks: first, Using recombinant plasmids pGEM-T-Rhipilin-1 and pGEM-T-Luciferase as templates, the target gene Rhipilin-1dsRNA and control gene Luciferase dsRNA; were synthesized in vitro from ticks according to the operating instructions of T7 kit. Secondly, the optimum lipids of ticks in each stage were screened, the soaking time and the ratio of dsRNA to lipids were selected, and the distribution of dsRNA was observed by the soaking of fluorescent labeled dsRNA. Thirdly, the optimum lipids, soaking time, and the proportion of soaking to juvenile ticks of falciform fan head tick, if tick and adult ticks were soaked in three stages, the dsRNA of Luciferase gene was used as control, and the animal experiment was carried out. The results showed that the optimal soaking conditions were as follows: Lipofectamine 2000, the volume ratio of dsRNA to lipids was 2: 1, and the volume ratio of lipids to lipids was 1: 1 if DMRIE-C, was soaked for 12 hours. The volume ratio of DMRIE-C, to lipids was 1: 2. Fluorescent dsRNA entered ticks. The saturated time and body weight of the marinated ticks were significantly increased, which had a significant effect on the original biological functions of the ticks. Therefore, Rhipilin-1 gene can be used as a candidate gene for tick-killing agents, which provides a basis for the application of RNA interference methodology in ticks control and the creation of new environmentally friendly ticks.
【學位授予單位】：南京農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S852.7

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