白纹伊蚊气味结合蛋白（OBP）基因的克隆和功能分析

发布时间：2018-05-22 08:24

本文选题：白纹伊蚊 + 吸血行为　；参考：《中国人民解放军军事医学科学院》2010年硕士论文

【摘要】： 白纹伊蚊Aedes albopictus是我国登革热和登革出血热的主要传播媒介,也是城市灭蚊的主要对象。目前,对白纹伊蚊进行化学防治一直是控制登革热流行的主要手段,然而,随着蚊虫抗药性的出现和抗药性水平的不断增高,防治效果受到影响,同时,化学杀虫剂对环境造成的污染也一直是无法避免的问题。因此,近年来,如何有效利用白纹伊蚊的生理行为特点,发展新的防治方法已日益受到关注。以往的研究发现,蚊虫对宿主的吸血习性是以嗅觉为基础,并由遗传基因所决定。蚊虫吸血行为的嗅觉机制中,多种蛋白参与了蚊虫对宿主气味分子的识别和定位过程。在这些蛋白中,气味结合蛋白(odorant-binding protein,OBP)数量最多,它位于触角感器的淋巴液中,直接与外界环境中成千上万的气味分子接触。气味分子一般为脂溶性的化合物,必须与OBP结合才能穿过亲水性的触角感器淋巴液,到达神经树突膜上的气味受体,产生动作电位。OBP识别和结合气味分子是蚊虫感受宿主气味的第一步,在蚊虫寻找血源定位宿主的过程中起重要作用。目前国内外对蚊虫OBP的研究主要集中在冈比亚按蚊上,也有少量关于埃及伊蚊、致倦库蚊和白纹伊蚊的报道,其中白纹伊蚊OBP的研究报道仅有3个基因序列被识别且没有更深入的研究。因此,白纹伊蚊的高度嗜人血习性可能与哪些OBP基因有关,又是哪些OBP基因参与了白纹伊蚊重复吸血的独特生理过程,这些都不清楚。而OBP基因作用于白纹伊蚊嗜血行为中感受宿主气味的第一步,因此找到这些与其吸血行为相关的OBP基因并明确其功能是揭示白纹伊蚊吸血行为嗅觉机制的突破口,对于阐明白纹伊蚊吸血习性的内在分子机制具有重要的意义。更重要的是,对白纹伊蚊吸血习性内在机制的阐明,也为发展新的防治方法干扰白纹伊蚊-人宿主之间的联系,从而阻断蚊媒病的流行提供了新的思路和途径。本研究结果如下: (1)通过构建白纹伊蚊雌蚊的触角cDNA文库,克隆得到2个气味结合蛋白核酸序列,分别与埃及伊蚊OBP 56a和56e具有大于80%的相似度,命名为AealOBP1和AealOBP2;另外还得到2个嗅觉受体、1个气味受体、1个味觉受体和3个荷尔蒙受体的基因核酸序列,都与埃及伊蚊相关基因的相似性最高。 (2)通过设计简并引物,对白纹伊蚊雌蚊触角cDNA进行扩增,得到6个OBP基因序列,其中1个与文库中发现的AealOBP1序列相重合,剩余5个OBP基因序列分别与埃及伊蚊、致倦库蚊和冈比亚按蚊OBP基因序列具有大于75%的相似性,命名为AealOBP3至AealOBP7; (3)实时定量PCR检测10个OBP(AealOBP1、AealOBP3～7、AealOBP46、AealOBP67和AealOBP68)基因在雌蚊吸血前后和雄蚊头部起始表达量的差异,其中AealOBP1、AealOBP4和AealOBP5基因在吸血前后雌蚊头部和雌雄蚊头部表达无显著差异;饱血雌蚊头部AealOBP6、AealOBP46和AealOBP68的表达量明显多于未吸血雌蚊头部,且AealOBP67和AealOBP46在雄蚊头部的表达量也明显多于未吸血雌蚊;AealOBP3和AealOBP7在吸血后雌蚊头部表达量显著降低,而AealOBP7在未吸血雌蚊头部的表达量又明显多于雄蚊。
[Abstract]:Aedes albopictus Aedes albopictus is the main medium of dengue and dengue hemorrhagic fever in China, and it is also the main object of urban mosquito control. At present, chemical control of Aedes albopictus has been the main means to control the epidemic of dengue fever. However, with the emergence of mosquito resistance and the increasing resistance level, the control effect has been affected. At the same time, the environmental pollution caused by chemical pesticides has also been an unavoidable problem. Therefore, in recent years, how to effectively use the physiological behavior of Aedes albopictus and the development of new control methods have been paid more and more attention.
Previous studies have found that the blood sucking habit of mosquitoes to the host is based on the olfactory sense and is determined by genetic genes. In the olfactory mechanism of the mosquito sucking behavior, a variety of proteins participate in the process of identification and localization of mosquitoes to host odorants. In these proteins, the number of odorant-binding protein, OBP is the most, and its number is the most. In the lymph fluid of the antennae, it is directly contacted with thousands of odorant molecules in the external environment. Odorant molecules are generally fat soluble compounds, which must be combined with OBP to pass through the hydrophilic tentacle lymph, to reach the odorant receptors on the nerve dendrite membrane, and to produce dynamic potential.OBP recognition and combination of odorant molecules that are mosquitoes. The first step of host odor plays an important role in the process of locating the host by the mosquito in search of the source of the source of the blood. At present, the study of mosquitoes at home and abroad is mainly concentrated in Anopheles Gambia, and a small amount of reports on Aedes aegypti, Culex pipiens and Aedes albopictus, of which only 3 gene sequences have been identified and only identified in the study of Aedes albopictus OBP. There is no more in-depth study. Therefore, the high eosinophilic habit of Aedes albopictus may be related to which OBP genes and which OBP genes participate in the unique physiological process of the Aedes albopictus in repeated bloodsucking, which is not clear. And the OBP gene acts on the first step of the Aedes albopictus to feel the host odor in the bloodthirsty behavior of Aedes albopictus. The OBP gene related to the blood sucking behavior and its function are a breakthrough in revealing the olfactory mechanism of Aedes albopictus, which is important to elucidate the intrinsic molecular mechanism of the blood sucking habit of Aedes albopictus, and more importantly, the internal mechanism of the blood sucking habit of Aedes albopictus, and the development of new control methods to interfere with the Aedes albopictus. The link between human hosts, thereby blocking the prevalence of mosquito borne diseases, has provided new ideas and approaches.
The results of this study are as follows:
(1) by constructing the antennae cDNA Library of Aedes albopictus, 2 odorant binding protein nucleic acid sequences were cloned, and the similarity between the OBP 56a and 56e of Aedes aegypti was greater than 80%, respectively, named AealOBP1 and AealOBP2, and 2 olfactory receptors, 1 odorant receptors, 1 taste receptors and 3 hormone receptor sequences were also obtained. All of them have the highest similarity to Aedes aegypti related genes.
(2) through the design of degenerate primers, 6 OBP gene sequences were amplified for the cDNA of Aedes albopictus, and 1 of them coincided with the AealOBP1 sequences found in the library. The remaining 5 OBP sequences were similar to the OBP gene sequences of Aedes aegypti, Culex pipiens tirenopus and Anopheles Gambia, respectively, named AealOBP3 to AealOB P7;
(3) the difference in the initial expression of 10 OBP (AealOBP1, AealOBP3 to 7, AealOBP46, AealOBP67 and AealOBP68) genes in the head of the male and male mosquitoes was detected by real time quantitative PCR. There was no significant difference in the expression of AealOBP1, AealOBP4 and AealOBP5 genes between the head of the female and male and male mosquitoes before and after the blood sucking; the head of the female mosquito satiety was AealOBP6, AealOBP46. The expression of and AealOBP68 was significantly more than the head of the non blood sucking female mosquitoes, and the expression of AealOBP67 and AealOBP46 in the head of the male mosquitoes was significantly more than that of the non blood sucking females. The expression of AealOBP3 and AealOBP7 in the head of the female mosquitoes decreased significantly after the blood sucking, and the expression of AealOBP7 in the head of the non blood sucking female mosquitoes was significantly more than that of the male mosquito.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R384.1

【引证文献】