保幼激素类似物Methoprene耐受基因1和G蛋白偶联受体激酶2在棉铃虫发育过程中的功能研究

发布时间：2018-03-30 04:11

  本文选题：棉铃虫　切入点：JH　出处：《山东大学》2016年博士论文

【摘要】：研究背景随着分子生物学技术的发展,人类研究昆虫发育的机制有了更好的平台。通过阐明昆虫发育的分子机理,可以为人类研究与疾病相关的生理过程提供参考和理论依据。全变态昆虫的整个生命周期要经历多次蜕皮,包括幼虫各龄期之间的蜕皮、幼虫向蛹过渡的变态蜕皮以及蛹到成虫的羽化蜕皮。昆虫的发育和蜕皮变态过程主要受蜕皮激素(20E)和保幼激素(JH)共同调控。因此,阐明20E膜信号通路终止机制和JH胞内受体Methoprene耐受基因1(Met1)传导JH信号的机制具有重大意义。国内外研究进展及科学问题目前关于昆虫JH功能的研究主要集中在对其受体功能机制的研究上,关于最有可能的JH受体Methoprene耐受基因(Met)的研究结果有很多。Met结合JH后和bHLH-PAS家族其他蛋白一起作为复合体结合到Krh1基因启动子区域JH响应元件(JHRE)中的E-Box上,启动Krhl的转录,进而传导JH信号；在赤拟谷盗中干扰Met,会引起幼虫的早熟变态。超气门蛋白(USP)参与20E信号通路的同时还能与JH结合。Met作为JH的胞内受体如何传导JH信号,USP如何与Met相互作用一起参与JH通路,这些科学问题的答案尚不明确。20E膜信号通路是目前的研究热点,20E最有可能的膜受体是GPCR。G蛋白偶联受体激酶(GRK)最著名的功能是磷酸化G蛋白偶联受体(GPCR),使GPCR信号脱敏。棉铃虫中的GRK2分布在细胞质中,GRK2如何在20E的诱导下由细胞质移位至细胞膜终止GPCR的功能,GRK2是否介导20E膜信号的终止都是目前研究的热点科学问题。研究结果本文以棉铃虫及其表皮细胞系为实验材料,通过体内和体外实验,运用分子生物学技术,深入研究了Met1和GRK2在棉铃虫生长发育中的功能及机制,获得如下结果和结论：1.磷酸化的Met1和非磷酸化USP1形成转录复合体促进基因转录维持幼虫状态Metl的表达被JH Ⅲ上调,但是被高浓度的20E抑制。虫体干扰Metl导致提前化蛹。Metl通过促进JH通路基因转录和抑制20E通路基因转录来维持幼虫状态。无激素时,存在Metl-Metl-USP1复合体；JHⅢ诱导下,Met1的PAC结构域发生磷酸化导致Metl同源二聚体解离,Metl和Hsp90,USP1一起以复合体形式结合到Krhl基因启动子中的.RE序列上起始Krhl的转录；在20E诱导下,USP1的第21位丝氨酸发生磷酸化,打破Metl与USP1的结合并破坏Metl-Met1的二聚化,Met1以单体形式存在,磷酸化的USP1与20E的核受体EcR-B1结合形成转录复合体传导20E信号。2.GRK2终止GPCR在细胞膜上传导20E信号的功能GRK2蛋白在虫体变态时期高表达,GRK2的表达被20E上调。在虫体干扰GRK2导致化蛹进程加速、20E通路基因转录上调和凋亡变态过程的提前。20E通过细胞膜上的ErGPCR-2调控PKC磷酸化GRK2的第680位丝氨酸,导致GRK2的膜转位；位于细胞膜上的GRK2结合ErGPCR-2并使之磷酸化导致ErGPCR-2的内吞,进而终止ErGPCR-2传导20E信号的功能。20E和GRK2之间存在负反馈调控机制。结论及意义Met1通过抑制20E通路并促进JH通路基因转录来维持幼虫状态,磷酸化的Met1和非磷酸化USP1一起传导JH信号,本文的研究结果为JH维持昆虫幼虫状态抑制变态发生提供了新的实验证据和理论支持。通过对GRK2的功能研究,揭示了类固醇激素20E终止的机制,阐明了GRK2和ErGPCR-2相互作用使20E通路在虫体中正常的发挥作用不致过量。为昆虫发育及蜕皮变态提供了新的理论知识,为害虫防治提供的新的靶标。
[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.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q966
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本文编号：1684291