青海血蜱水通道蛋白（AQPs）基因亚型鉴定与生物学功能分析

Tick is obligate hematophagous ectoparasite in vertebrates, it is the second largest biological vector for many pathogens. Blood sucking and transmission of diseases have harmed livestock industry and human health. The prevention and control of tick have become the focus of attention and at home and abroad. The control strategies for ticks mainly depend on the application of chemicals. However, intensive use of chemicals could result in resistance and environmental contamination. The alternative control strategy is the development of the new preparation of anti-tick. There are a lot of activities of liquid transport during transmission of pathogen and life cycle in ticks. Aquaporins (AQPs) are important functional proteins and widely present in cell membrane of almost all organisms, mediating transmembrane transport of liquid. The present studies have proved that AQPs play an important role in fluid transport. Therefore, it may be as a key protein against tick, but there are a very few studies internationally on this aspect regarding AQPs in tick, the transportation mechanism remains unclear. Haemaphysalis qinghaiensis is the dominant tick species in the northwest farmland of China, and it is responsible for transmission of various pathogens of ruminant livestock. In this study, based on the partial sequence of AQPs obtained from constructed cDNA library of Haemaphysalis qinghaiensis, we will use the biology research methods, including molecular biology, bioinformatics and RNA interference and so on, to systematically probe the subtypes, the distribution of AQPs expression in Haemaphysalis qinghaiensis, protein structure and biological function of AQPs etc. The results could be provided evidences for the development of new drugs and anti-tick vaccines. This study has important theoretical significance for revealing the mechanism of liquid transport in ticks, as well as practical application value for tick control.

蜱是专性吸血的外寄生虫，是传播多种人和动物疾病的第二大生物媒介，给人类健康和畜牧业带来极大危害。蜱的防控是近年国内外关注的焦点和研究热点。化学药物控制蜱存在抗药性和环境污染等缺陷，新型抗蜱制剂具有广泛的应用前景。蜱叮咬宿主传播病原及生活周期中存在大量液体运输活动，水通道蛋白（AQPs）是广泛存在生物体细胞膜上一类专性介导液体跨膜转运的重要功能蛋白，已证明在液体运输中发挥关键作用，可能作为控制蜱的关键蛋白，但蜱体内AQPs研究甚少，生物学功能也不清楚。青海血蜱是仅在我国分布并传播多种病原，对畜牧业危害最严重的蜱种之一。本研究在前期构建的青海血蜱cDNA文库中扩增出AQPs部分序列的基础上，运用分子生物学、生物信息学和RNA干扰等技术，对AQPs的亚型、种类、表达分布、蛋白结构及生物学功能等进行系统研究。为新型抗蜱药物或疫苗的研制提供新的理论依据，对揭示蜱体内液体运输机制和蜱防治具有重要意义。

蜱是专性吸血的外寄生虫，是传播多种人和动物疾病的第二大生物媒介，给人类健康和畜牧业带来极大危害。蜱的防控是近年国内外关注的焦点和研究热点。化学药物控制蜱存在抗药性和环境污染等缺陷，新型抗蜱制剂具有广泛的应用前景。蜱叮咬宿主传播病原及生活周期中存在大量液体运输活动，水通道蛋白（AQPs）是广泛存在生物体细胞膜上一类专性介导液体跨膜转运的重要功能蛋白，已证明在液体运输中发挥关键作用，可能作为控制蜱的关键蛋白，但蜱体内AQPs研究甚少，生物学功能也不清楚。青海血蜱是仅在我国分布并传播多种病原，对畜牧业危害最严重的蜱种之一。. 本研究按照计划及研究预期目标，通过克隆测序后得到了4种不同的青海血蜱AQP基因的全长序列，运用blastn和序列比对软件分析，确定其为青海血蜱AQP基因异构体。通过氨基酸序列比对分析，发现仅C端序列存在差异，其余氨基酸序列完全一致。系统进化树分析发现青海血蜱四种AQP基因序列与非洲扇头蜱和血红扇头蜱的AQP序列同源性较高（98%），初步认定其为不同的青海血蜱AQP基因的转录异构体; 利用在线软件分析AQP生物学信息，预测其生物学功能，发现青海血蜱AQP均有6个跨膜区，无信号肽序列，疏水性较强; 采用亲水性标签，利用无细胞表达系统成功表达了AQP蛋白; 合成了青海血蜱AQP三条外膜环多肽段，并制备了多克隆抗体; 提取青海血蜱不同发育阶段和组织器官的总RNA，应用定量Q-PCR检测了AQP基因表达变化。