【作者】 张俊英；

【导师】 赵小凡；

【作者基本信息】 山东大学 ， 生物化学与分子生物学， 2018， 硕士

【摘要】 研究背景及科学问题昆虫是地球上数量最多、分布最广的动物群落。昆虫的生长发育主要由保幼激素和蜕皮激素(20-羟基蜕皮酮,20E)协同调控。在昆虫取食生长时期,虫体内保幼激素滴度高,而在昆虫蜕皮变态时期,保幼激素逐渐降低直至零,蜕皮激素滴度逐渐增加。两种激素存在相互作用,既拮抗又统一,协同调控昆虫的蜕皮与变态过程。研究已证明20E存在着经典的基因组信号通路,20E通过自由扩散,或在细胞膜受体介导下,跨越细胞膜进入细胞内,与核受体EcR结合起始基因的转录表达。近些年,科学研究陆续发现了 20E的非基因组途径。例如胞内蛋白的快速磷酸化及移位,胞内钙离子动员等。也有研究发现G蛋白偶联受体(GPCR)参与蜕皮激素的非基因组信号途径,而不同的GPCR的作用机制各异。因此GPCR参与20E的非基因组信号通路的问题成为人们的关注热点。本实验室已证明ErGPCR1、ErGPCR2参与20E的非基因组信号通路。在果蝇中发现多巴胺受体(DmDopEcR)也可以结合20E的类似物[3H]]PonA,但是多巴胺受体在20E信号通路中的作用机制尚不清楚。本论文以棉铃虫为实验材料开展了相关研究,探究DopEcR在20E信号通路中的作用及机制。结果及结论DopEcR在棉铃虫幼虫的不同组织均有表达,包括表皮、中肠、脂肪体和脑,且在脑中的表达量最高,在蜕皮及变态期的脑和中肠中有升高的趋势。20E促进DopEcR的表达。DopEcR参与调控20E诱导的胞内钙离子水平的快速增加、基因表达及昆虫的变态过程。DopEcR定位于细胞膜,且20E诱导下不发生内吞。RNA干扰DopEcR后降低了 20E与细胞膜的结合水平,过表达DopEcR后增加了 20E与细胞膜的结合水平,纯化后的DopEcR能与20E发生结合,但是其结合位点突变体与20E的结合能力显著降低。我们的研究表明20E上调DopEcR的表达,DopEcR通过结合20E调控昆虫的变态发育。研究的新发现及意义:本论文以生化与分子生物学的技术方法进行研究,针对GPCR动态结合20E的特性,设计建立了新的方法,首次在鳞翅目昆虫中证明了 DopEcR可以结合20E,为鉴定GPCR结合其它配体提供了借鉴。该研究阐明了 DopEcR主要分布在脑,说明了组织分布不同可能是多个GPCR参与20E信号途径的机制之一。本研究首次报道类固醇激素20E可以促进神经系统的分子DopEcR表达,而DopEcR又参与20E途径调控昆虫取食及发育,说明20E与多巴胺系统存在正反馈调控关系,共同调控昆虫生长发育。该研究丰富和完善了蜕皮激素调控昆虫生长发育的理论知识,为20E的非基因组信号途径提供了新的实验证据,为害虫防治提供了新的靶标基因。更多还原

【Abstract】 BackgroundInsects are the largest and most widely distributed animal community on earth.The growth and development of insects are mainly regulated by the conteractive regulation of juvenile hormone(JH)and 20-hydroxyecdysone(20E).During the growth period of insect feeding,the titer of JH is high and the 20E is low.However,the JH is gradually decresed or until zero and the 20E is gradually increased during the metamorphosis of insects.The two kinds of hormones are antagonistic and unified to regulate the molting and metamorphosis of insects.Researches prove steroid hormones exist classical genomic pathway.20E can across the plasma membrane and enter cells through free diffusion,and bind nuclear receptors to start gene transcription.In recent years,researches discover the nongenomic pathway of 20E,such as rapid phosphorylation and displacement of intracellular proteins,intracellular calcium mobilization.It is also found that G-protein-coupled receptors(GPCRs)involve in the nongenomic pathway of steroid hormone.And different GPCRs have different mechanisms.Therefore,the studies of involvenent of GPCRs in the nongenomic pathway of 20E become a focus of attention.Our laboratory has demonstrated that ErGPCRl and ErGPCR 2 are involved in the nongenomic pathway of 20E.Some dopamine receptor(DopEcR)is also involved in the signaling of 20F.But the mechanism is unclear.This paper carried out related research on Helicoverpa armigera to explore the mechanism of DopEcR in regulating the nongenomic pathway of 20E.Results and ConclusionHere,using a lepidopteran insect H.armigera as a model,DopEcR was expressed in different orgnization,including epidermis,midgut,fat body,and in the highest amount of expression in brain.DopEcR has a tendency to rise in brain and midgut at molting and metamorphic molting.DopEcR was upregulated by 20E.DopEcR was involved in 20E-induced rapid increase of intracellular calcium ion,gene expression and insect metamorphosis.DopEcR was located on the plasma membrane without internalization by 20E exciting.Knockdown of DopEcR by RNA interference reduced the binding of 20E to plasma membrane.Overexpression of DopEcR increased the binding of 20E to plasma membrane.DopEcR could bind 20E after being isolated;however,the binding-site mutant of DopEcR decreased capacity to bind 20E.Our study suggested 20E upregulates DopEcR expression for transmitting 20E signal to regulate insect development.Highlights and significanceThe binding of DopEcR and 20E was demonstrated by a new method of laboratory discussion in addition to the basic research of the existing technical methods in the laboratory.This method provides reference for the identification of other ligands and receptors.And this study improved and enriched the theoretical knowledge of insect growth and development and the signal pathway of 20E.It provides a theoretical basis for the prevention and treatment of natural insects.更多还原