长角血蜱唾液腺功能蛋白发掘与吸血适应机制研究

发布时间：2018-03-29 07:49

本文选题：长角血蜱　切入点：转录组　出处：《河北师范大学》2017年硕士论文

【摘要】：蜱是一种专性吸血的外寄生节肢动物,可携带并传播多种病原体,造成人、畜患上疾病甚至死亡,严重影响人类健康和畜牧业发展。长角血蜱Haemaphysalis longicornis隶属于硬蜱科Ixodidae血蜱属Haemaphysalis,该物种分布广泛,数量众多,危害性极大。长角血蜱为完成整个生活史需要吸食大量宿主血液,其中唾液腺作为重要器官,可分泌多种促进吸血和消化的活性分子,帮助长角血蜱完成正常的吸血活动。研究唾液腺不同吸血时期蛋白差异表达对于阐明长角血蜱的唾液腺吸血的分子机制,进而全面揭示长角血蜱的生存适应对策具有重要意义。本实验利用第二代测序技术对长角血蜱雌蜱饥饿期、交配前吸血期、交配后半饱期和饱血期的四个吸血阶段进行转录组测序。测序并合并组装,共产生87825条Unigene,其中长度在1Kb以上的Unigene有13222条,进行功能注释,共注释29109条Unigene。利用该转录组序列作为数据库,进行长角血蜱唾液腺4个不同吸血时期所有蛋白质的定量蛋白质组学研究。最终通过基于iTRAQ的定量蛋白质组学技术,以长角血蜱4个不同吸血时期的唾液腺蛋白作为研究对象,鉴定到表达差异蛋白3223条,其中具有注释信息的蛋白3164条,未注释蛋白59条。利用生物信息学方法对差异表达蛋白进行系统分析,最终发现了众多与调控促进吸血蛋白表达和运输相关的蛋白,我们挑选了其中一些重要蛋白进行了RNA干扰,例如驱动蛋白、动力蛋白、网格蛋白、分选蛋白、半乳糖激酶和腺苷酰硫酸激酶,最终发现驱动蛋白和动力蛋白被干扰后,雌蜱吸血受到明显抑制,导致其过早从宿主体表脱落甚至死亡。通过功能分析,我们推测两种蛋白在长角血蜱吸血功能分子运输过程中起着重要作用。本论文利用定量蛋白质组学方法对长角血蜱不同吸血时期唾液腺蛋白进行研究,并结合研究结果筛选到能明显影响雌蜱吸血的两种蛋白----驱动蛋白和动力蛋白,这两种蛋白也是首次在长角血蜱中得到报道。实验结果对于阐明长角血蜱唾液腺生理变化的分子机制以及长角血蜱适应吸血的分子机制具有重要意义,对于通过分子手段抑制蜱类吸血具有一定的指导意义,并为蜱类的综合防治提供理论依据。
[Abstract]:Tick is a specialized blood sucking parasite arthropod, can carry and spread a variety of pathogens, causing human, animal disease or even death, Haemaphysalis longicornis belongs to the genus Haemaphysalis, which belongs to the genus Haemaphysalis of the family Ixodidae. The species is widely distributed, abundant and harmful. Haemaphysalis longicornis needs to take a lot of host blood in order to complete the whole life cycle of Haemaphysalis. The salivary gland, as an important organ, can secrete a variety of active molecules that promote blood sucking and digestion. To help Haemaphysalis longicornis to complete normal blood sucking activities. To study the molecular mechanism of salivary gland blood sucking of Haemaphysalis longicornis by studying the differential expression of protein in different blood sucking stages of salivary gland. Therefore, it is of great significance to reveal the survival and adaptation strategies of Haemaphysalis longicornis. The second generation sequencing technique was used to study the feeding period and the blood sucking stage of Haemaphysalis longicornii. After mating, four transcriptome sequences were sequenced in half full period and full blood stage. The sequence was combined and assembled to produce 87825 Unigenees, of which 13222 Unigene with length above 1Kb were annotated. This transcriptional sequence was used as a database to study the quantitative proteomics of all proteins in the salivary glands of Haemaphysalis longicornis at four different stages of bloodsucking. Finally, the quantitative proteomics technique based on iTRAQ was used. The salivary gland proteins of Haemaphysalis longicornis at four different stages were identified as expressing 3223 differentially expressed proteins, among which 3164 were annotated. 59 unannotated proteins. The differential expression proteins were systematically analyzed by bioinformatics, and many proteins related to regulating and promoting the expression and transport of blood-sucking proteins were found. We selected some of the important proteins for RNA interference, such as proteins such as driving protein, dynamic protein, grid protein, sorting protein, galactose kinase and adenosine sulfate kinase, and finally found that the protein and dynamic protein were interfered. Female ticks' blood sucking is significantly inhibited, resulting in premature abscission or even death from the host's body surface. We speculate that two proteins play an important role in the molecular transport of Haemaphysalis longicornis. In this paper, the salivary gland proteins of Haemaphysalis longicornis were studied by quantitative proteomics. Combined with the results of the study, two kinds of proteins, which can significantly affect the blood absorption of female ticks, were screened out. These two proteins were also reported for the first time in Haemaphysalis longicornis. The experimental results are of great significance in elucidating the molecular mechanism of the physiological changes of salivary glands of Haemaphysalis longicornis and the molecular mechanisms of Haemaphysalis longicornis adapting to blood sucking. It has certain guiding significance for the inhibition of ticks' blood sucking by molecular means, and provides the theoretical basis for the comprehensive control of ticks.
【学位授予单位】：河北师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.746;R384.4

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