本文選題：TRIC-B　切入點：20E　出處：《山東大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：研究背景、存在的科學(xué)問題級研究意義昆蟲是世界上品類、個體數(shù)量最豐富的動物類群,對人類在農(nóng)業(yè)、醫(yī)學(xué)等領(lǐng)域的生產(chǎn)活動有著巨大影響。圍繞對害蟲的防治,多以昆蟲作為模型研究多種生理生化現(xiàn)象機理。昆蟲包括完全變態(tài)昆蟲,其發(fā)育過程經(jīng)歷卵、幼蟲、蛹、成蟲和不完全變態(tài)昆蟲,其發(fā)育過程經(jīng)歷卵、幼蟲變態(tài)發(fā)育成成蟲。蛻皮(molting)和變態(tài)(metamorphosis)是昆蟲的生長發(fā)育中必須經(jīng)歷的兩個過程。昆蟲蛻皮變態(tài)過程主要受蛻皮激素20羥基蛻皮酮(20-hydroxyecdysone,20E)及保幼激素(Juvenile Hormone,JH)調(diào)控,其中蛻皮發(fā)生由20E起始,蛻皮的性質(zhì)由JH決定。經(jīng)典的20E基因組途徑認(rèn)為20E直接進入細(xì)胞并啟動下游基因的轉(zhuǎn)錄,而近年來的研究表明:某些G蛋白偶聯(lián)受體(G-protein-coupledreceptor,GPCR)存在于細(xì)胞膜上,能夠響應(yīng)蛻皮激素信號,并介導(dǎo)細(xì)胞內(nèi)的鈣離子濃度升高。靜息狀態(tài)下,細(xì)胞內(nèi)鈣離子貯存在細(xì)胞內(nèi)質(zhì)網(wǎng)(endoplasmic reticulum,ER)中;激素刺激下,細(xì)胞內(nèi)鈣離子由內(nèi)質(zhì)網(wǎng)上的儲存調(diào)控型鈣離子通道(store-operated calcium channel,SOC)釋放到細(xì)胞質(zhì)。SOC是非神經(jīng)興奮性細(xì)胞中的鈣通道,受細(xì)胞信號刺激產(chǎn)生三磷酸肌醇(inositol 1,4,5-triphosphate,IP3),IP3 通過 IP3 受體(inositol 1,4,5-triphosphate receptor,IP3R)介導(dǎo)將內(nèi)質(zhì)網(wǎng)貯存的鈣離子釋放到細(xì)胞質(zhì)。細(xì)胞外鈣離子由受體調(diào)控的鈣離子通道內(nèi)流進入細(xì)胞質(zhì)。由于鈣離子是帶正電荷的陽離子,因而內(nèi)質(zhì)網(wǎng)內(nèi)儲存的鈣離子釋放到細(xì)胞質(zhì)的過程引起內(nèi)質(zhì)網(wǎng)膜內(nèi)外電荷不平衡。細(xì)胞內(nèi)需要其他離子,如鉀離子、氯離子和氫離子等作為平衡離子補充鈣離子快速釋放引起的內(nèi)質(zhì)網(wǎng)膜電位失衡(Fink and Veigel 1996;NAGASAWA et al.1984)。三聚體胞內(nèi)陽離子通道蛋白(Trimeric intracellular cation channel,TRIC)是一種存在于細(xì)胞內(nèi)質(zhì)網(wǎng)/肌質(zhì)網(wǎng)(ER/SR)并附著在核膜上的陽離子通道蛋白,在細(xì)胞中主要參與內(nèi)質(zhì)網(wǎng)鉀離子雙向運輸。TRIC作為雙向鉀離子通道參與平衡細(xì)胞內(nèi)鈣離子釋放導(dǎo)致的內(nèi)質(zhì)網(wǎng)電位差。TRIC蛋白通道包含兩個亞家族:TRIC-A和TRIC-B。其中TRIC-A大量存在于可興奮性細(xì)胞中,響應(yīng)蘭尼堿受體介導(dǎo)的內(nèi)質(zhì)網(wǎng)/肌質(zhì)網(wǎng)鈣離子釋放;TRIC-B少量但廣泛地存在于各種細(xì)胞中,響應(yīng)IP3R介導(dǎo)的內(nèi)質(zhì)網(wǎng)鈣離子釋放。既然20E能夠誘導(dǎo)IP3R開放,而TRIC-B又可以響應(yīng)IP3R介導(dǎo)的鈣離子釋放,那么TRIC-B是否參與蛻皮激素20E信號途徑?研究報道TRIC-B通道參與發(fā)育過程(Yamazaki et al.2009),其機制如何?目前尚無研究報道。本文以鱗翅目害蟲棉鈴蟲(Halicoverpa armigera)作為研究材料,并利用實驗室前期建立的棉鈴蟲表皮細(xì)胞系(HaEpi)為平臺,研究了 TRIC-B參與調(diào)節(jié)20E誘導(dǎo)的鈣離子釋放和內(nèi)流過程,及其對昆蟲發(fā)育的影響,并在20E在生長過程中對鈣離子的調(diào)節(jié)機理方面有所促進,為防治害蟲提供了新的靶標(biāo)。研究結(jié)果及結(jié)論本實驗以棉鈴蟲為實驗對象,通過分子生物學(xué)技術(shù)、生物化學(xué)及細(xì)胞生物學(xué)技術(shù),研究三聚體胞內(nèi)陽離子通道蛋白B(TRIC-B)在昆蟲生長發(fā)育過程中的作用,并取得以下結(jié)果:TRIC-B在蛻皮期高表達;干擾TRIC-B導(dǎo)致幼蟲發(fā)育失敗死亡;TRIC-B通過維持內(nèi)質(zhì)網(wǎng)電荷平衡介導(dǎo)20E誘導(dǎo)的細(xì)胞內(nèi)鈣釋放和外鈣內(nèi)流;TRIC-B定位于細(xì)胞質(zhì);干擾TRIC-B導(dǎo)致HaEpi細(xì)胞死亡。結(jié)論:TRIC-B作為離子平衡通道介導(dǎo)20E調(diào)控的內(nèi)質(zhì)網(wǎng)中鈣離子的有效釋放及細(xì)胞外鈣離子內(nèi)流。TRIC-B在棉鈴蟲發(fā)育過程和細(xì)胞存活中起關(guān)鍵性作用,缺失TRIC-B幼蟲和細(xì)胞發(fā)生死亡。研究的創(chuàng)新點及意義TRIC-B能夠維持20E誘導(dǎo)的鈣離子釋放及細(xì)胞外鈣離子內(nèi)流過程。豐富了蛻皮激素20E非基因組途徑的理論,驗證了 TRIC-B通道在生長發(fā)育過程中的重要作用,為害蟲控制提供了良好靶標(biāo)。
[Abstract]:The research background, research significance of insect science problem is the existence of the world category, animal taxa were most abundant in the number of individuals, to human beings in agriculture, medicine and other fields have a huge impact in the production activities. The control of pests, to study a variety of insects as a physiological phenomenon. The mechanism model of insects including holometabolous insects the developmental process of experience, egg, larva, pupa, adult and incomplete metamorphosis development experience, eggs, metamorphosis in larvae develop into adult worms. Molting and metamorphosis (molting) (metamorphosis) is the two process of insect growth and development must be experienced. Molt and metamorphosis process is mainly affected by molting hormone 20 HYDROXYECDYSONE (20-hydroxyecdysone, 20E) and juvenile hormone (Juvenile, Hormone, JH) regulation, which occurs by molting 20E initiation, molting properties determined by the JH. The 20E gene group pathway that 20E directly into the fine The cell and activate its downstream gene transcription, but recent studies have shown that certain G protein coupled receptors (G-protein-coupledreceptor, GPCR) on the cell membrane, in response to ecdysone signal, and mediated by the intracellular calcium concentration increased. The resting state, intracellular calcium stored in the endoplasmic reticulum (endoplasmic reticulum. ER); hormone stimulation, intracellular calcium regulation by storage type calcium channel Er (store-operated calcium channel, SOC).SOC is released into the cytoplasm calcium channel non nerve excitability in the cells by cell signaling stimulates the production of inositol phosphate (inositol three 1,4,5-triphosphate, IP3), IP3 (via IP3 receptor inositol 1,4,5-triphosphate receptor, IP3R) mediated endoplasmic reticulum calcium storage and release into the cytoplasm. Extracellular calcium by calcium channel receptor regulation within the flow into the fine Due to the cytoplasm. The calcium ion is positively charged cations, Ca2 + and ER storage is released into the cytoplasm of process induced endoplasmic reticulum and charge imbalance. Cells need other ions, such as potassium, calcium ion balance as of the rapid release of endoplasmic reticulum membrane potential caused by the imbalance of chloride ion and hydrogen ion (such as Fink and Veigel 1996; NAGASAWA et al.1984). The trimer of intracellular cation channel protein (Trimeric intracellular cation channel, TRIC) is a present in the endoplasmic reticulum / sarcoplasmic reticulum (ER/SR) and attached cation channel protein in the nuclear membrane, the cells mainly involved in endoplasmic reticulum potassium ion transport bidirectional.TRIC as the two potassium channels involved in balance of intracellular Ca2 + release of endoplasmic reticulum potential difference in the protein.TRIC pathway contains two subfamilies: TRIC-A and TRIC-B. which can exist in the TRIC-A Excitatory cells in response to endoplasmic reticulum / ryanodine receptor mediated sarcoplasmic reticulum calcium release; a small amount of TRIC-B but is widely used in all kinds of cells in response to endoplasmic reticulum calcium mediated release of IP3R. Since 20E can induce the opening of IP3R, while TRIC-B can respond to calcium mediated release of IP3R. That TRIC-B is involved in ecdysone signal pathway of 20E research reports? TRIC-B channels are involved in the development process (Yamazaki et al.2009), what is the mechanism? There is no research. This paper reports to Lepidoptera pests of cotton bollworm (Halicoverpa armigera) as the research materials, and the use of cotton bollworm epidermal cell lines the pre established (HaEpi) platform. Study of TRIC-B involved in the regulation of calcium release induced by 20E and flow process, and its effect on insect development, and in the 20E in the process of growth regulating mechanism of calcium ions on surface was enhanced, in order to prevent Provide a new target for treatment of pests. The results and conclusions of this experiment to cotton bollworm as the experimental object, through the technology of molecular biology, biochemistry and cell biology technology research, trimeric intracellular cation channel protein B (TRIC-B) in the process of growth and development of insects in the role, and achieved the following results: TRIC-B in high molting period the expression of TRIC-B in larval development; interference failure death; TRIC-B by maintaining the endoplasmic reticulum charge balance mediated 20E induced intracellular calcium release and calcium influx; TRIC-B located in the cytoplasm; TRIC-B interference induced HaEpi cell death. Conclusion: in the process of the development of cotton bollworm and plays a key role in cell survival.TRIC-B release and extracellular calcium ion balance as TRIC-B channel mediated regulation of 20E in the endoplasmic reticulum in the death of larvae and deletion of TRIC-B cells. The innovation of research and the significance of TRIC-B The process flow of calcium release and extracellular calcium to maintain 20E induced. Enriched 20E ecdysone non genomic signaling theory, verifies the important role of TRIC-B channels in the process of development and growth, provide a good target for pest control.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S433
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本文編號：1566713