本文選題：棉鈴蟲　切入點(diǎn)：JH　出處：《山東大學(xué)》2016年博士論文

【摘要】：研究背景隨著分子生物學(xué)技術(shù)的發(fā)展,人類研究昆蟲發(fā)育的機(jī)制有了更好的平臺(tái)。通過闡明昆蟲發(fā)育的分子機(jī)理,可以為人類研究與疾病相關(guān)的生理過程提供參考和理論依據(jù)。全變態(tài)昆蟲的整個(gè)生命周期要經(jīng)歷多次蛻皮,包括幼蟲各齡期之間的蛻皮、幼蟲向蛹過渡的變態(tài)蛻皮以及蛹到成蟲的羽化蛻皮。昆蟲的發(fā)育和蛻皮變態(tài)過程主要受蛻皮激素(20E)和保幼激素(JH)共同調(diào)控。因此,闡明20E膜信號(hào)通路終止機(jī)制和JH胞內(nèi)受體Methoprene耐受基因1(Met1)傳導(dǎo)JH信號(hào)的機(jī)制具有重大意義。國內(nèi)外研究進(jìn)展及科學(xué)問題目前關(guān)于昆蟲JH功能的研究主要集中在對(duì)其受體功能機(jī)制的研究上,關(guān)于最有可能的JH受體Methoprene耐受基因(Met)的研究結(jié)果有很多。Met結(jié)合JH后和bHLH-PAS家族其他蛋白一起作為復(fù)合體結(jié)合到Krh1基因啟動(dòng)子區(qū)域JH響應(yīng)元件(JHRE)中的E-Box上,啟動(dòng)Krhl的轉(zhuǎn)錄,進(jìn)而傳導(dǎo)JH信號(hào)；在赤擬谷盜中干擾Met,會(huì)引起幼蟲的早熟變態(tài)。超氣門蛋白(USP)參與20E信號(hào)通路的同時(shí)還能與JH結(jié)合。Met作為JH的胞內(nèi)受體如何傳導(dǎo)JH信號(hào),USP如何與Met相互作用一起參與JH通路,這些科學(xué)問題的答案尚不明確。20E膜信號(hào)通路是目前的研究熱點(diǎn),20E最有可能的膜受體是GPCR。G蛋白偶聯(lián)受體激酶(GRK)最著名的功能是磷酸化G蛋白偶聯(lián)受體(GPCR),使GPCR信號(hào)脫敏。棉鈴蟲中的GRK2分布在細(xì)胞質(zhì)中,GRK2如何在20E的誘導(dǎo)下由細(xì)胞質(zhì)移位至細(xì)胞膜終止GPCR的功能,GRK2是否介導(dǎo)20E膜信號(hào)的終止都是目前研究的熱點(diǎn)科學(xué)問題。研究結(jié)果本文以棉鈴蟲及其表皮細(xì)胞系為實(shí)驗(yàn)材料,通過體內(nèi)和體外實(shí)驗(yàn),運(yùn)用分子生物學(xué)技術(shù),深入研究了Met1和GRK2在棉鈴蟲生長發(fā)育中的功能及機(jī)制,獲得如下結(jié)果和結(jié)論：1.磷酸化的Met1和非磷酸化USP1形成轉(zhuǎn)錄復(fù)合體促進(jìn)基因轉(zhuǎn)錄維持幼蟲狀態(tài)Metl的表達(dá)被JH Ⅲ上調(diào),但是被高濃度的20E抑制。蟲體干擾Metl導(dǎo)致提前化蛹。Metl通過促進(jìn)JH通路基因轉(zhuǎn)錄和抑制20E通路基因轉(zhuǎn)錄來維持幼蟲狀態(tài)。無激素時(shí),存在Metl-Metl-USP1復(fù)合體；JHⅢ誘導(dǎo)下,Met1的PAC結(jié)構(gòu)域發(fā)生磷酸化導(dǎo)致Metl同源二聚體解離,Metl和Hsp90,USP1一起以復(fù)合體形式結(jié)合到Krhl基因啟動(dòng)子中的.RE序列上起始Krhl的轉(zhuǎn)錄；在20E誘導(dǎo)下,USP1的第21位絲氨酸發(fā)生磷酸化,打破Metl與USP1的結(jié)合并破壞Metl-Met1的二聚化,Met1以單體形式存在,磷酸化的USP1與20E的核受體EcR-B1結(jié)合形成轉(zhuǎn)錄復(fù)合體傳導(dǎo)20E信號(hào)。2.GRK2終止GPCR在細(xì)胞膜上傳導(dǎo)20E信號(hào)的功能GRK2蛋白在蟲體變態(tài)時(shí)期高表達(dá),GRK2的表達(dá)被20E上調(diào)。在蟲體干擾GRK2導(dǎo)致化蛹進(jìn)程加速、20E通路基因轉(zhuǎn)錄上調(diào)和凋亡變態(tài)過程的提前。20E通過細(xì)胞膜上的ErGPCR-2調(diào)控PKC磷酸化GRK2的第680位絲氨酸,導(dǎo)致GRK2的膜轉(zhuǎn)位；位于細(xì)胞膜上的GRK2結(jié)合ErGPCR-2并使之磷酸化導(dǎo)致ErGPCR-2的內(nèi)吞,進(jìn)而終止ErGPCR-2傳導(dǎo)20E信號(hào)的功能。20E和GRK2之間存在負(fù)反饋調(diào)控機(jī)制。結(jié)論及意義Met1通過抑制20E通路并促進(jìn)JH通路基因轉(zhuǎn)錄來維持幼蟲狀態(tài),磷酸化的Met1和非磷酸化USP1一起傳導(dǎo)JH信號(hào),本文的研究結(jié)果為JH維持昆蟲幼蟲狀態(tài)抑制變態(tài)發(fā)生提供了新的實(shí)驗(yàn)證據(jù)和理論支持。通過對(duì)GRK2的功能研究,揭示了類固醇激素20E終止的機(jī)制,闡明了GRK2和ErGPCR-2相互作用使20E通路在蟲體中正常的發(fā)揮作用不致過量。為昆蟲發(fā)育及蛻皮變態(tài)提供了新的理論知識(shí),為害蟲防治提供的新的靶標(biāo)。
[Abstract]:Background: with the development of molecular biology technology, better platform mechanism of human development. Through the study of insects to elucidate the molecular mechanism of insect development, can provide reference and theoretical basis for the study of human physiological processes associated with the disease. The insect of the whole life cycle to undergo ecdysis, including different instar larvae the larvae molt, molting and metamorphosis of transition to pupa pupa to adult eclosion molt. Development and metamorphosis of insect molting is mainly affected by ecdysone and juvenile hormone (20E) (JH) Co regulation. Therefore, to clarify the 20E membrane receptor Methoprene gene signaling termination tolerance mechanism and intracellular JH (Met1) is 1 the significance of the transmission mechanism of JH signal. The domestic and foreign research progress and scientific problems of current research on insect JH function mainly focused on the study of its receptor function mechanism, on the can JH receptor Methoprene gene (Met) can withstand the results there are a lot of.Met combined with JH and other bHLH-PAS family proteins together as a complex binding to the promoter region of Krh1 gene JH response element (JHRE) in E-Box, the transcription of Krhl, JH and Met in signal conduction; interference caused by larvae in Tribolium. The precocious metamorphosis. Ultraspiracle (USP) involved in 20E signaling pathway can also be combined with JH.Met JH as an intracellular receptor to JH transduction, how USP interacts with Met in the JH pathway, the scientific answer to the question is not clear.20E membrane signaling pathway is the research hotspot at present, membrane receptor 20E the most likely is a GPCR.G protein coupled receptor kinase (GRK) is the most famous feature is the phosphorylation of G protein coupled receptor (GPCR), the GPCR signal desensitization. The distribution of GRK2 in cotton bollworm in the cytoplasm, GRK2 in 20E Induced from the cytoplasm to the cell membrane translocation to terminate the GPCR function, GRK2 termination 20E mediated membrane signal is a hot scientific problem in the present study. The results of this study to cotton bollworm epidermal cell line as the experimental material, through in vivo and in vitro experiments, using molecular biology technology, in-depth study of the function and mechanism of growth in the development of Met1 and GRK2 in the cotton bollworm, get the following results and conclusions: 1. the expression of phosphorylated Met1 and non phosphorylated USP1 transcription complex formation promoting gene transcription by JH Metl larvae maintain state III increases, but the high concentration of 20E inhibited. Worm Metl interference leads to early pupation.Metl by promoting JH pathway inhibition of 20E gene transcription and gene transcription pathway to maintain the larval state. No hormone, Metl-Metl-USP1 complex; JH III induced by PAC domain Met1 phosphorylation leads to homologous Metl Two dimer dissociation, Metl and Hsp90, USP1 in the form of complex binding to Krhl gene promoter.RE sequence in the initiation of transcription of Krhl; induced by 20E, phosphorylation of USP1 at serine twenty-first, breaking the combination of Metl and USP1 and destroy the dimerization of Metl-Met1, Met1 in monomer form, combined with USP1 and 20E phosphorylation of the nuclear receptor EcR-B1 is highly expressed in insect metamorphosis during the formation of transcription complex 20E transduction.2.GRK2 termination GPCR conduction in the cell membrane of the 20E signal function of GRK2 protein, the expression of GRK2 was up-regulated by 20E interference of GRK2 resulted in the insect body. Pupation accelerated in early.20E 20E pathway up-regulated and apoptosis metamorphosis through the membrane of the ErGPCR-2 regulation of PKC phosphorylation of GRK2 at serine 680th, resulting in membrane translocation of GRK2; located on the cell membrane of GRK2 combined with ErGPCR-2 and the phosphorylation of ErGPCR-2 in lead Swallow, there is a negative feedback regulation mechanism between ErGPCR-2 and 20E signal conduction termination function of.20E and GRK2. The conclusion and significance of Met1 by inhibiting the 20E pathway and JH pathway to promote gene transcription to maintain the larval state, phosphorylation of Met1 and non phosphorylated USP1 with JH signal transmission, the results of this study for JH to maintain state inhibition of insect larvae abnormal occurrence provides new experimental evidence and theoretical support. Through the study of GRK2 functions, reveals the mechanism of steroid hormone 20E termination, expounds the interaction between ErGPCR-2 and GRK2 20E pathway in the body in normal play a role not excessive. Provide new knowledge for the development of insects and insect metamorphosis, provide the new target for pest control.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q966
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本文編號(hào)：1684291