青海4种皮蝇蛆病病原分类及差异蛋白组学研究

发布时间：2018-01-11 05:17

  本文关键词：青海4种皮蝇蛆病病原分类及差异蛋白组学研究　出处：《西北农林科技大学》2016年博士论文　论文类型：学位论文

  更多相关文章： 皮蝇蛆病 物种鉴定 系统发育 群体遗传 同工酶 蛋白质组

【摘要】：皮蝇蛆病(Hypodermosis)是双翅目(Diptera)、环裂亚目(Cyclorrhapha)、有瓣类(Calyptratae)、皮蝇科(Hypodermatidae)的不同阶段的幼虫感染多种动物和人的一种寄生虫病。其中在我国报道的致黄牛和牦牛皮蝇蛆病的皮蝇主要有牛皮蝇(H.bovis)、纹皮蝇(H.lineatum)和中华皮蝇(H.sinense),致藏羚羊皮蝇蛆病的皮蝇主要有藏羚羊?皮蝇(Pr.orongonis)和藏羚羊皮蝇(H.pantholopsum),致高原鼠兔皮蝇蛆病的皮蝇主要有兔裸皮蝇(O.leporina)、异裸皮蝇(O.prodigiosa)和轲裸皮蝇(O.koslowi)。目前青海黄牛、牦牛、藏羚羊和高原鼠兔感染皮蝇蛆病病原在分类上还存在一定争议,本研究从黄牛、牦牛、藏羚羊和高原鼠兔感染皮蝇蛆病病原三期幼虫微观形态、分子生物学和同工酶理化特性等方面进行综合评价,以期完善青海4种皮蝇蛆病病原分类;同时采用蛋白质组和质谱技术研究了青海4种皮蝇蛆病病原三期幼虫差异蛋白生物功能及蛋白表达调控的各自特点,获得了以下研究结果:1.青海4种皮蝇蛆三期幼虫种类鉴定及超微形态学观察(1)从形态学和分子生物学两个方面对青海4种皮蝇蛆进行了种类鉴定,结果表明青海4种皮蝇蛆为牛皮蝇(H.bovis)、中华皮蝇(H.sinense)、藏羚羊皮蝇(H.pantholopsum)和兔裸皮蝇(O.leporina)4个独立的种。(2)通过扫描电镜形态观察,详细的描述了牛皮蝇、中华皮蝇、藏羚羊皮蝇和兔裸皮蝇三期幼虫具有关键鉴别部位的伪头、口孔、第10节腹面以及气门板各不相同的微观形态。2.青海4种皮蝇蛆系统发育和起源时间分析(1)基于COI数据集,测序获得了60个牛皮蝇、52个中华皮蝇、14个藏羚羊皮蝇和25个兔裸皮蝇线粒体COI基因部分序列,分别得到17个、23个、12个和21个单倍型;综合贝叶斯法系统发生分析和Beast分化时间的估算,进一步证实了牛皮蝇、中华皮蝇、藏羚羊皮蝇和兔裸皮蝇是4个有效的独立种。(2)AMOVA检测发现牛皮蝇和中华皮蝇群体间在各自地理区域存在显著的遗传结构差异(P0.001),并且群体内的分化高于群体间的分化;地理结构分析发现牛皮蝇和中华皮蝇由于生境喜好和扩散能力的差异而导致群体遗传结构的不同;种群历史动态分析发现牛皮蝇和中华皮蝇种群经历了突然扩张事件,主要都在晚更新世(late Pleistocene)期间发生的扩张。3.青海4种皮蝇蛆乳酸脱氢酶(LDH)和酯酶(EST)比较研究(1)青海4种皮蝇蛆LDH和EST同工酶电泳结果显示,4种皮蝇LDH和EST酶谱的表达形式和浓度各不相同,具有一定种属特异性,说明牛皮蝇、中华皮蝇、藏羚羊皮蝇和兔裸皮蝇是4个独立种。(2)酶活性检测发现,4种皮蝇蛆LDH最适pH均为6.0,牛皮蝇、中华皮蝇和藏羚羊皮蝇EST在pH 9.0时最稳定,兔裸皮蝇EST在pH 8.5时最稳定;4种皮蝇蛆LDH和EST最适反应温度均为55℃。4.青海4种皮蝇蛆病病原差异蛋白组分析(1)牛皮蝇、中华皮蝇、藏羚羊皮蝇和兔裸皮蝇三期幼虫蛋白质表达图谱上分别检测到399、378、363和359个蛋白点;利用MALDI-TOF-MS质谱鉴定,结合MASCOT、Uniprot等数据库检索及文献资料共鉴定出56个差异蛋白质,这些蛋白质主要参与碳水化合物代谢、蛋白质代谢、能量代谢、分子转运、应激反应、发育调控、细胞骨架等生物过程。(2)差异蛋白质表达量聚类分析表明,17个蛋白质在牛皮蝇高表达,其中有1个细胞骨架蛋白,2个蛋白质代谢调控蛋白,3个信号调节蛋白,2个能量代谢调控蛋白,2个应激反应蛋白,1个催化反应蛋白,1个分子转运蛋白,2个蛋白质生物合成蛋白,2个发育蛋白和1个防御反应蛋白;21个蛋白质在中华皮蝇高表达,其中有2个碳水化合物代谢蛋白,1个细胞骨架蛋白,2个发育蛋白,2个催化反应蛋白,1个蛋白质代谢调控蛋白,2个蛋白质生物合成蛋白,2个能量代谢调控蛋白,2个应激反应蛋白,2个免疫反应蛋白和5个信号调节蛋白;18个蛋白质在藏羚羊皮蝇高表达,其中有1个分子转运蛋白,1个免疫反应蛋白,3个信号调节蛋白,2个催化反应蛋白,2个应激反应蛋白,4个细胞骨架蛋白,1个碳水化合物代谢蛋白,1个能量代谢调控蛋白,1个蛋白质生物合成蛋白,1个发育蛋白和1个蛋白质代谢调控蛋白。9个蛋白质在兔裸皮蝇高表达,其中有2个能量代谢调控蛋白,3个细胞骨架蛋白,1个蛋白质代谢调控蛋白,1个信号调节蛋白,1个催化反应蛋白和1个蛋白质生物合成蛋白。综上所述,本研究通过种类鉴定、系统发育、分化时间以及同工酶比较,明确了青海4种皮蝇蛆病病原为牛皮蝇、中华皮蝇、藏羚羊皮蝇和兔裸皮蝇,并详细描述了青海省4种皮蝇蛆三期幼虫具有关键鉴别部位的微观形态,可为今后皮蝇种的形态鉴别以及更好的完善皮蝇种微观形态特征资料提供一定的理论参考;还对青海4种皮蝇蛆某些关键蛋白的定性和功能分析,可以从整体的视角了解青海4种皮蝇蛆病病原在生长发育过程中的调控机制。这些发现丰富了皮蝇蛆病病原研究数据,可为今后青藏高原皮蝇蛆病流行病学监测和控制提供理论依据。
[Abstract]:Hypodermosis is (Hypodermosis) (Diptera), Diptera cyclorrhapha (Cyclorrhapha), calyptratae (Calyptratae), HYPODERMATIDAE (Hypodermatidae) a parasitic disease of different stages of larvae infected with a variety of animal and human. The main hypodermabovis in China reported by cattle and yak Hypodermosis the Hypoderma (H.bovis), Hypoderma lineatum (H.lineatum) and the hypoderma.sp (H.sinense), Hypoderma caused by Tibetan antelope Hypodermosis mainly Tibetan antelope? Hypoderma.sp (Pr.orongonis) and Tibetan antelope hypoderma.sp (H.pantholopsum), hypoderma.sp induced plateau pika Hypodermosis mainly rabbit pelt flies (O.leporina). Different pelt flies (O.prodigiosa) and Ke pelt flies (O.koslowi). At present, Qinghai cattle, yak, Tibetan antelope and plateau pika infection pathogen Hypodermosis there are still some controversy in the classification, this study from the cattle, yak, Tibetan antelope and plateau pika Hypodermosis pathogen infection three Larval morphology, isozyme and molecular biology for comprehensive evaluation of physicochemical characteristics and other aspects, in order to improve the Qinghai 4 kinds of pathogenic myiasis classification; their characteristics were also studied in Qinghai of 4 kinds of pathogenic myiasis in three stage larvae protein biological function and protein expression regulation by proteomics and mass spectrometry, obtained the following results: observation the three species identification and ultrastructural morphology of the 1. Qinghai 4 seed maggot larvae (1) were identified from two aspects of morphology and molecular biology of Qinghai 4 seed fly, the results show that the Qinghai 4 skin for maggot hypodermabovis (H.bovis), Chinese hypoderma.sp (H.sinense), Tibetan antelope hypoderma.sp (H.pantholopsum) and rabbit naked hypoderma.sp (O.leporina) 4 independent species. (2) by scanning electron microscopy morphology, a detailed description of the Hypoderma lineatum, the Tibetan antelope, flies and flies three rabbit pelt larvae with the The key parts of the differential pseudo head hole, analysis of Tenth ventrite and valve plate microstructure of.2. of different Qinghai 4 seed maggot phylogeny and the origin of the time (1) based on COI data sets, we obtained 60 hypodermabovis, 52 Chinese hypoderma.sp, part of the 14 Tibetan antelope and 25 flies rabbit pelt flies mitochondrial COI gene sequences, respectively 17, 23, 12 and 21 haplotypes; analysis and estimation of Beast differentiation time integrated Bias Fa system occurred, further confirmed the hypoderma, the Tibetan antelope skin flies, flies and flies rabbit pelt are 4 effective independent species (2. AMOVA) detected hypodermabovis and Chinese hypoderma.sp groups in their respective geographical areas have significant differences in genetic structure (P0.001), and the differentiation within population was higher than that of differentiation among populations; and the analysis found that the Hypoderma lineatum due to differences in habitat preferences and diffusion ability of geography structure Due to the different genetic structure; found hypodermabovis and the hypoderma.sp population experienced a sudden expansion of population dynamic history event analysis, mainly in the late Pleistocene (late Pleistocene) during the expansion of the.3. Qinghai 4 seed maggot lactic dehydrogenase (LDH) and esterase (EST) Comparative Study (1) Qinghai 4 seed maggot LDH and EST electrophoresis results showed that the expression and concentration of 4 kinds of flies LDH and EST isozymes vary, with some species specificity, indicating the Hypoderma lineatum, Tibetan antelope, flies and flies rabbit pelt are 4 separate species. (2) found that the detection of enzyme activity, 4 kinds of maggot LDH, the optimum pH was 6, the Hypoderma lineatum, Tibetan antelope and fly EST the most stable at pH 9. The rabbit pelt flies EST the most stable at pH 8.5; LDH and EST 4 kinds of maggot optimum reaction temperature is 55 DEG.4. Qinghai 4 seed proteins were analysis of pathogenic myiasis (1) The hypoderma, hypoderma, Tibetan antelope flies and rabbit pelt flies three stage larvae protein expression profiles were detected on the 399378363 and 359 protein spots were identified by mass spectrometry; MALDI-TOF-MS, combined with MASCOT, Uniprot database retrieval and literature identified a total of 56 different proteins, these proteins are mainly involved in the regulation of carbohydrate metabolism. Protein metabolism, energy metabolism, molecular transport, stress response, cytoskeleton development and other biological processes. (2) the difference of protein expression of cluster analysis showed that 17 proteins highly expressed in hypodermabovis, of which there are 1 cytoskeletal proteins, 2 protein metabolic regulation protein 3 signal regulated protein, 2 energy metabolic regulation protein, 2 stress proteins, 1 catalytic protein, 1 molecular transporters, 2 protein biosynthesis protein, 2 protein and 1 Developmental defense reaction protein; 21 protein High quality in the expression of hypoderma.sp, of which there are 2 carbohydrate metabolism 1 proteins, cytoskeletal proteins, 2 developmental protein, 2 catalytic protein, 1 protein metabolic regulation protein, 2 protein biosynthesis protein, 2 protein and energy metabolism, stress response protein 2, 2 immune response the 5 signal protein and regulatory protein; 18 proteins highly expressed in the Tibetan antelope flies, of which there are 1 molecular transporters, 1 immunoreactive proteins, 3 signal regulated protein, 2 protein catalytic reaction, 2 stress response proteins, 4 cell skeleton protein, 1 carbohydrate metabolism proteins, 1 an energy metabolism protein, 1 protein biosynthesis protein, 1 developmental proteins and 1 protein metabolism regulation protein.9 protein expression in rabbit pelt flies, of which there are 2 energy metabolism proteins, 3 cell skeleton protein, 1 protein metabolism 1 signal regulatory proteins, regulatory proteins, 1 catalytic proteins and 1 protein biosynthesis protein. In summary, this study through species identification, phylogeny, comparative differentiation time and isozyme, clear Qinghai 4 kinds of myiasis pathogen Hypoderma lineatum, the Tibetan antelope, flies and flies with rabbit pelt, and describes the morphology of Qinghai province 4 seed maggot three stage larvae are the key parts of the differential, which can provide a theoretical reference for the future of Hypoderma species identification and improve the micro morphological characteristics of hypoderma.sp data; qualitative and function of Qinghai 4 seed maggot protein analysis of some key, from the overall perspective to understand the regulation mechanism of 4 kinds of pathogens of Qinghai in the process of development and growth. These findings enrich Hypodermosis pathogenic research data, for the future of Tibetan Plateau disease epidemic maggot skin The theoretical basis is provided for the monitoring and control of the study.

【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S855.9
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本文编号：1408257