Rhipilin-1基因双链RNA对镰形扇头蜱抑制及杀灭效果

发布时间：2018-11-12 06:46

【摘要】：蜱是常见的人和动物体表寄生虫,作为嗜血性节肢动物,蜱在分类学上属于节肢动物门、蛛形纲、蜱螨目、蜱总科,其生活史复杂,繁殖率高,经历卵、幼虫、若虫、成虫四个发育阶段,属于不完全变态发育。作为多种人兽共患病病原的储存宿主和传播媒介,蜱可传播和感染病原引发原虫病、森林脑炎、新疆出血热、回归热、莱姆病、Q热、立克次体病等。威胁人类健康,对畜牧经济发展带来巨大危害。因此,预防和控制蜱的传播十分重要。镰形扇头蜱属于硬蜱的一种,广泛分布于我国长江以南,是我国南方的优势蜱种。目前对杀蜱除蜱的主要方法是化学药物杀灭,这种方法对蜱的杀伤效果虽好,但环境污染严重,毒性药物对宿主危害性大,随着生物技术研究的深入,新型环保的预防控制蜱的策略不断发展,如抗蜱疫苗研究等,但还未取得成功,新的防治技术需求还十分迫切。RNA干扰技术作为新兴的研究技术,在蜱研究中得到广泛应用。本实验室已克隆鉴定了镰形扇头蜱抗凝血分子Rhipilin-1,并发现通过显微注射证明RNA干扰该基因可有效降低蜱的吸血能力。本研究根据RNA干扰原理,将Rhipilin-1dsRNA直接浸泡蜱体,通过脂质体浸染传递途径达到RNA干扰效果,从而实施如杀虫剂灭蜱的新型防治技术:其一,以重组质粒pGEM-T-Rhipilin-1和pGEM-T-Luciferase为模板,根据T7试剂盒的操作说明,在蜱体外合成目的基因Rhipilin-1dsRNA和对照基因Luciferase dsRNA;其二,在浸泡过程中筛选蜱各阶段最佳脂质剂、浸泡时间以及dsRNA与脂质剂浸泡比例并通过荧光标记dsRNA的浸泡观察dsRNA分布;其三,以最佳脂质剂、浸泡时间、浸泡比例对镰形扇头蜱幼蜱、若蜱和成蜱三个阶段浸泡,以Luciferase基因的dsRNA作为对照,进行动物体实验观察。结果显示,最佳浸泡条件筛选为幼蜱脂质剂Lipofectamine 2000、浸泡24h、dsRNA与脂质剂体积比为2:1;若蜱脂质剂DMRIE-C、浸泡12h、dsRNA与脂质剂体积比为1:1;幼蜱脂质剂DMRIE-C、浸泡12h、dsRNA与脂质剂体积比为1:2;荧光dsRNA进入蜱体;浸泡后的镰形扇头蜱饱血时间显著增长,饱血体重显著减少,即对镰形扇头蜱原有生物学功能产生显著影响。因此,可将Rhipilin-1基因作为杀蜱剂的候选基因,这为RNA干扰方法学在蜱防控实践应用以及新型环保的杀蜱剂的创制提供基础。
[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.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.7

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