本文關(guān)鍵詞：青海4種皮蠅蛆病病原分類及差異蛋白組學(xué)研究　出處：《西北農(nóng)林科技大學(xué)》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 皮蠅蛆病 物種鑒定 系統(tǒng)發(fā)育 群體遺傳 同工酶 蛋白質(zhì)組

【摘要】：皮蠅蛆病(Hypodermosis)是雙翅目(Diptera)、環(huán)裂亞目(Cyclorrhapha)、有瓣類(Calyptratae)、皮蠅科(Hypodermatidae)的不同階段的幼蟲(chóng)感染多種動(dòng)物和人的一種寄生蟲(chóng)病。其中在我國(guó)報(bào)道的致黃牛和牦牛皮蠅蛆病的皮蠅主要有牛皮蠅(H.bovis)、紋皮蠅(H.lineatum)和中華皮蠅(H.sinense),致藏羚羊皮蠅蛆病的皮蠅主要有藏羚羊?皮蠅(Pr.orongonis)和藏羚羊皮蠅(H.pantholopsum),致高原鼠兔皮蠅蛆病的皮蠅主要有兔裸皮蠅(O.leporina)、異裸皮蠅(O.prodigiosa)和軻裸皮蠅(O.koslowi)。目前青海黃牛、牦牛、藏羚羊和高原鼠兔感染皮蠅蛆病病原在分類上還存在一定爭(zhēng)議,本研究從黃牛、牦牛、藏羚羊和高原鼠兔感染皮蠅蛆病病原三期幼蟲(chóng)微觀形態(tài)、分子生物學(xué)和同工酶理化特性等方面進(jìn)行綜合評(píng)價(jià),以期完善青海4種皮蠅蛆病病原分類;同時(shí)采用蛋白質(zhì)組和質(zhì)譜技術(shù)研究了青海4種皮蠅蛆病病原三期幼蟲(chóng)差異蛋白生物功能及蛋白表達(dá)調(diào)控的各自特點(diǎn),獲得了以下研究結(jié)果:1.青海4種皮蠅蛆三期幼蟲(chóng)種類鑒定及超微形態(tài)學(xué)觀察(1)從形態(tài)學(xué)和分子生物學(xué)兩個(gè)方面對(duì)青海4種皮蠅蛆進(jìn)行了種類鑒定,結(jié)果表明青海4種皮蠅蛆為牛皮蠅(H.bovis)、中華皮蠅(H.sinense)、藏羚羊皮蠅(H.pantholopsum)和兔裸皮蠅(O.leporina)4個(gè)獨(dú)立的種。(2)通過(guò)掃描電鏡形態(tài)觀察,詳細(xì)的描述了牛皮蠅、中華皮蠅、藏羚羊皮蠅和兔裸皮蠅三期幼蟲(chóng)具有關(guān)鍵鑒別部位的偽頭、口孔、第10節(jié)腹面以及氣門(mén)板各不相同的微觀形態(tài)。2.青海4種皮蠅蛆系統(tǒng)發(fā)育和起源時(shí)間分析(1)基于COI數(shù)據(jù)集,測(cè)序獲得了60個(gè)牛皮蠅、52個(gè)中華皮蠅、14個(gè)藏羚羊皮蠅和25個(gè)兔裸皮蠅線粒體COI基因部分序列,分別得到17個(gè)、23個(gè)、12個(gè)和21個(gè)單倍型;綜合貝葉斯法系統(tǒng)發(fā)生分析和Beast分化時(shí)間的估算,進(jìn)一步證實(shí)了牛皮蠅、中華皮蠅、藏羚羊皮蠅和兔裸皮蠅是4個(gè)有效的獨(dú)立種。(2)AMOVA檢測(cè)發(fā)現(xiàn)牛皮蠅和中華皮蠅群體間在各自地理區(qū)域存在顯著的遺傳結(jié)構(gòu)差異(P0.001),并且群體內(nèi)的分化高于群體間的分化;地理結(jié)構(gòu)分析發(fā)現(xiàn)牛皮蠅和中華皮蠅由于生境喜好和擴(kuò)散能力的差異而導(dǎo)致群體遺傳結(jié)構(gòu)的不同;種群歷史動(dòng)態(tài)分析發(fā)現(xiàn)牛皮蠅和中華皮蠅種群經(jīng)歷了突然擴(kuò)張事件,主要都在晚更新世(late Pleistocene)期間發(fā)生的擴(kuò)張。3.青海4種皮蠅蛆乳酸脫氫酶(LDH)和酯酶(EST)比較研究(1)青海4種皮蠅蛆LDH和EST同工酶電泳結(jié)果顯示,4種皮蠅LDH和EST酶譜的表達(dá)形式和濃度各不相同,具有一定種屬特異性,說(shuō)明牛皮蠅、中華皮蠅、藏羚羊皮蠅和兔裸皮蠅是4個(gè)獨(dú)立種。(2)酶活性檢測(cè)發(fā)現(xiàn),4種皮蠅蛆LDH最適pH均為6.0,牛皮蠅、中華皮蠅和藏羚羊皮蠅EST在pH 9.0時(shí)最穩(wěn)定,兔裸皮蠅EST在pH 8.5時(shí)最穩(wěn)定;4種皮蠅蛆LDH和EST最適反應(yīng)溫度均為55℃。4.青海4種皮蠅蛆病病原差異蛋白組分析(1)牛皮蠅、中華皮蠅、藏羚羊皮蠅和兔裸皮蠅三期幼蟲(chóng)蛋白質(zhì)表達(dá)圖譜上分別檢測(cè)到399、378、363和359個(gè)蛋白點(diǎn);利用MALDI-TOF-MS質(zhì)譜鑒定,結(jié)合MASCOT、Uniprot等數(shù)據(jù)庫(kù)檢索及文獻(xiàn)資料共鑒定出56個(gè)差異蛋白質(zhì),這些蛋白質(zhì)主要參與碳水化合物代謝、蛋白質(zhì)代謝、能量代謝、分子轉(zhuǎn)運(yùn)、應(yīng)激反應(yīng)、發(fā)育調(diào)控、細(xì)胞骨架等生物過(guò)程。(2)差異蛋白質(zhì)表達(dá)量聚類分析表明,17個(gè)蛋白質(zhì)在牛皮蠅高表達(dá),其中有1個(gè)細(xì)胞骨架蛋白,2個(gè)蛋白質(zhì)代謝調(diào)控蛋白,3個(gè)信號(hào)調(diào)節(jié)蛋白,2個(gè)能量代謝調(diào)控蛋白,2個(gè)應(yīng)激反應(yīng)蛋白,1個(gè)催化反應(yīng)蛋白,1個(gè)分子轉(zhuǎn)運(yùn)蛋白,2個(gè)蛋白質(zhì)生物合成蛋白,2個(gè)發(fā)育蛋白和1個(gè)防御反應(yīng)蛋白;21個(gè)蛋白質(zhì)在中華皮蠅高表達(dá),其中有2個(gè)碳水化合物代謝蛋白,1個(gè)細(xì)胞骨架蛋白,2個(gè)發(fā)育蛋白,2個(gè)催化反應(yīng)蛋白,1個(gè)蛋白質(zhì)代謝調(diào)控蛋白,2個(gè)蛋白質(zhì)生物合成蛋白,2個(gè)能量代謝調(diào)控蛋白,2個(gè)應(yīng)激反應(yīng)蛋白,2個(gè)免疫反應(yīng)蛋白和5個(gè)信號(hào)調(diào)節(jié)蛋白;18個(gè)蛋白質(zhì)在藏羚羊皮蠅高表達(dá),其中有1個(gè)分子轉(zhuǎn)運(yùn)蛋白,1個(gè)免疫反應(yīng)蛋白,3個(gè)信號(hào)調(diào)節(jié)蛋白,2個(gè)催化反應(yīng)蛋白,2個(gè)應(yīng)激反應(yīng)蛋白,4個(gè)細(xì)胞骨架蛋白,1個(gè)碳水化合物代謝蛋白,1個(gè)能量代謝調(diào)控蛋白,1個(gè)蛋白質(zhì)生物合成蛋白,1個(gè)發(fā)育蛋白和1個(gè)蛋白質(zhì)代謝調(diào)控蛋白。9個(gè)蛋白質(zhì)在兔裸皮蠅高表達(dá),其中有2個(gè)能量代謝調(diào)控蛋白,3個(gè)細(xì)胞骨架蛋白,1個(gè)蛋白質(zhì)代謝調(diào)控蛋白,1個(gè)信號(hào)調(diào)節(jié)蛋白,1個(gè)催化反應(yīng)蛋白和1個(gè)蛋白質(zhì)生物合成蛋白。綜上所述,本研究通過(guò)種類鑒定、系統(tǒng)發(fā)育、分化時(shí)間以及同工酶比較,明確了青海4種皮蠅蛆病病原為牛皮蠅、中華皮蠅、藏羚羊皮蠅和兔裸皮蠅,并詳細(xì)描述了青海省4種皮蠅蛆三期幼蟲(chóng)具有關(guān)鍵鑒別部位的微觀形態(tài),可為今后皮蠅種的形態(tài)鑒別以及更好的完善皮蠅種微觀形態(tài)特征資料提供一定的理論參考;還對(duì)青海4種皮蠅蛆某些關(guān)鍵蛋白的定性和功能分析,可以從整體的視角了解青海4種皮蠅蛆病病原在生長(zhǎng)發(fā)育過(guò)程中的調(diào)控機(jī)制。這些發(fā)現(xiàn)豐富了皮蠅蛆病病原研究數(shù)據(jù),可為今后青藏高原皮蠅蛆病流行病學(xué)監(jiān)測(cè)和控制提供理論依據(jù)。
[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.

【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S855.9
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本文編號(hào)：1408257