昆虫蜕皮激素受体及其类似物的杀虫机制研究进展

昆虫的蜕皮、变态和繁殖受到蜕皮激素的严格调控。蜕皮激素作用靶标由蜕皮激素受体（ecdysteroid receptor, EcR）和超气门蛋白（ultraspiracle protein, USP）组成,蜕皮激素与EcR/USP作用启动蜕皮级联反应过程。昆虫EcR具有种类或类群的特异性,研究其结构、功能和调控机理在开发环境友好型新药剂和基因调控开关等方面具有重要指导作用。该文介绍了昆虫EcR的结构和功能特点,蜕皮激素及其类似物与EcR/USP的分子作用方式,以及基于EcR/USP的新杀虫剂创制和基因调控开关设计等方面的重要进展。     

斜纹夜蛾酵母双杂交文库的构建及其在蜕皮激素受体互作蛋白筛选中的应用

【目的】蜕皮激素在昆虫变态发育中起着关键的调控作用。蜕皮激素活化为20-羟基蜕皮酮(20-hydroxyecdysone,20E)后与蜕皮激素受体二聚体〔蜕皮激素受体(ecdysone receptor,EcR)和超气门蛋白(ultraspiracle protein,USP)〕结合,启动20E诱导的级联反应。本研究旨在从互作蛋白角度探究EcR/USP自身调控的分子机理。【方法】以重要农业害虫斜纹夜蛾Spodoptera litura为研究对象,通过构建酵母双杂交筛选系统分别筛选了与EcR和USP相互作用的蛋白。【结果】文库转化效率达到3.0×106cfu/μg,文库滴度达到1.3×108cfu/m L,文库插入片段大小在500~2 000 bp之间。4种诱饵质粒(EcRA/p GBKT7,EcRB1/p GBKT7,USP1/p GBKT7和USP2/p GBKT7)对酵母细胞无毒性,并且无自激活性,说明构建的酵母双杂交文库质量可靠。用以上4种诱饵质粒筛选酵母双杂交文库,共得到110个互作蛋白,其中与EcRB1,USP1和USP2互作的蛋白分别有26,52和32个,未筛选到与EcRA互作的蛋白。随后,从中挑选了Dna J-5(Hsp40 homolog 5,一种热激蛋白分子伴侣),MBF2(mediator of Bm FTZ-F1 type 2,一种转录共激活子),polyubiquitin(多聚泛素类蛋白),esr16(ecdysteroid regulated 16k Da protein,一种蜕皮激素调控蛋白)和NEDD8-like(neural precursor cell expressed,developmentally down-regulated protein 8,一种泛素调节相关蛋白)5种蛋白,利用酵母双杂交和Far-Western印迹法进一步验证了蛋白间的互作关系。【结论】分子伴侣和泛素化修饰等在蜕皮激素受体调控中可能起着重要作用。本研究对深入理解昆虫变态发育的分子机理具有重要意义。     

昆虫蜕皮激素信号转导途径研究进展

蜕皮与变态是全变态昆虫典型的发育特征。调控昆虫蜕皮与变态的激素主要有蜕皮激素和保幼激素。目前已经阐明了蜕皮激素的核受体EcR及部分核信号转导途径,但蜕皮激素是否存在膜受体及膜信号转导途径研究很少。研究证明,蜕皮激素存在细胞质中的信号转导分子和途径,蜕皮激素通过NTF2和Ran调控EcR入核启动基因转录。蜕皮激素使细胞质中的热休克蛋白Hsc70部分入核与USP结合启动基因转录。蜕皮激素通过蛋白激酶PKC使伴侣蛋白calponin磷酸化,参与蜕皮激素信号途径的基因转录。这些研究结果说明蜕皮激素除了有核受体和核受体信号转导途径外,还存在细胞膜受体和细胞膜信号转导途径。     

昆虫蜕皮激素受体研究进展

昆虫的蜕皮激素(molting hormone,MH)是甾醇类激素,在昆虫体内的活性形式为20-羟基蜕皮酮(20-hydroxyecdysone,20E)。在昆虫的正常发育过程中,昆虫的蜕皮、变态和繁殖受到蜕皮激素的调控。昆虫蜕皮激素受体(ecdysone receptor,EcR)和超气门蛋白(ultraspiracle,USP)均属于核受体超家族成员,具有核受体的结构特征,包括A/B域(转录激活域transactivation domain)、C域(DNA结合域DNA-binding domain,DBD)、D域(铰链域hinge region)、E域(配体结合域ligand binding domain,LBD)和F域。蜕皮激素受体在昆虫蜕皮、变态和繁殖等重要的生命过程中的级联反应启动位置,对昆虫的生长发育和繁殖的正常完成有着非常重要的作用。蜕皮激素通过与蜕皮激素受体和超气门蛋白组成的复合体相互作用,然后启动一系列级联反应的过程。本文介绍了EcR和USP的结构和功能,以及它们与蜕皮激素相互作用的机理,并对EcR在农业害虫防治等方面的应用进行了介绍,并讨论了研究中遇到的问题以及对未来的研究进行展望。     

灭幼脲作用机制的研究进展

<正> 灭幼脲(Diflubenzuron,TH-6040)是一类高效低毒并有一定选择性、而又比较广谱的新型杀虫剂。因为它的杀虫效应比较慢,为区别于普通杀虫剂,有人提议称之为制虫剂。关于它对昆虫的作用机制,目前还不很清楚,国内近年虽有不少有关综述,但一般只认为,灭幼脲主要作用于昆虫的体壁,抑制表皮几丁质的合成,使昆虫的蜕皮变态受阻而死亡。尽管有不少工作证明灭幼脲是抑制了几丁质合成酶     

抑食肼对槐尺蠖和丝绵木金星尺蠖的杀虫效果

在室内条件下测定了抑食肼对槐尺蠖和丝绵木金星尺蠖的杀虫效果,结果表明：（1）抑食肼对槐尺蠖和丝绵木金星尺蠖有良好的胃毒和触杀活性．同神经毒剂敌敌畏相比,抑食肼的速效性稍差,但持效性好。（2）抑食肼柜食迅速,处理12～24小时后幼虫取食被抑制,同时出现头壳剥离．（3）组织病理检查证明：抑食肼可诱导丝绵术金星尺蠖5龄幼虫出现皮质溶离,其作用类似于蜕皮激素。     

尺蠖蜕皮激素受体基因Eo-EcR 的克隆、生物信息学分析和表达检测

【目的】蜕皮激素受体（ecdysteroid receptor, EcR）是一种超家族核受体,它能与超气门蛋白USP组成异源二聚体复合物EcR/USP,调节20 羟基蜕皮酮（20E）的生物活性,进而调控昆虫的发育、变态及繁殖等生命过程。本研究旨在克隆茶尺蠖 Ectropis obliqua Prout EcR基因全长,并了解该基因的编码蛋白特征和时空表达模式。【方法】通过RT-PCR方法并结合RACE技术,克隆茶尺蠖EcR的基因全长,通过生物信息学软件和在线网站分析茶尺蠖EcR的生物学特性,通过实时荧光定量PCR（real-time quantitative PCR, qRT-PCR）技术比较茶尺蠖 EcR 在不同发育时期和6龄幼虫不同组织中的相对表达含量。【结果】克隆并鉴定了茶尺蠖EcR基因,将其命名为 Eo-EcR（基因登录号: KP869130.1）,Eo-EcR全长2 268 bp,含有1 728 bp开放阅读框,编码576个氨基酸。系统进化树和氨基酸同源性比对表明,Eo-EcR具有相对保守的进化特性,特别是与鳞翅目昆虫的保守性最高。三级结构模拟和功能结构域预测表明,Eo-EcR具有3个经典的结构模型,并以α螺旋为主,功能位点单一且为C₄型锌指结构。qRT-PCR结果表明,Eo-EcR在5龄和6龄幼虫期以及成虫期表达量较高,在其他龄期表达量变化不大;同时在前胸腺表达量最高,在血淋巴表达量最低。【结论】明确了Eo-EcR的核苷酸序列及编码蛋白特征,明确了Eo-EcR的时空表达特性。该研究结果为进一步研究Eo-EcR的分子功能和基于Eo-EcR为靶标杀虫剂的研制奠定分子基础。     

两种虫酰肼类新化合物对五种鳞翅目害虫的生物活性

为了比较虫酰肼及其衍生物对鳞翅目 5 种害虫的毒力 ,以虫酰肼为对照药剂,分别采用浸叶法、浸虫法和点滴法测定了虫酰肼衍生物 0593 (Ⅲ f) 和 0673 对甜菜夜蛾 Spodoptera exigua（Hübner）、斜纹夜蛾 Prodenia litura (Fabricius)、小菜蛾 Plutella xylostella (Linnaeus)、棉铃虫 Helicoverpa armigera(Hübner)和玉米螟 Ostrinia furnacalis(Guenée)的毒力;通过浸叶法系统研究了虫酰肼和虫酰肼衍生物 0593 对甜菜夜蛾生长发育的影响。结果表明：0593 和 0673 对甜菜夜蛾 4 龄幼虫的触杀毒力分别是虫酰肼的 11.3 倍和 7.4 倍,对斜纹夜蛾 4 龄幼虫触杀毒力分别是虫酰肼的 30.4 倍和 24.7 倍,对小菜蛾 3 龄幼虫浸虫处理的毒力分别是虫酰肼的 4.7 倍和 4.5 倍 ,对棉铃虫3龄幼虫的触杀毒力分别是虫酰肼的 15.5 倍和 15.2 倍,对玉米螟 4 龄幼虫的触杀毒力分别是虫酰肼的 1.3 倍和 2.0 倍,较虫酰肼的毒力都有很大提高,而0593 和 0673 对 5 种害虫的毒力差异不显著。用虫酰肼和 0593 处理甜菜夜蛾 2 龄幼虫,都能导致存活幼虫陆续死亡;化蛹率和正常蛹数降低;成虫羽化率、单雌产卵量和卵的孵化率显著减少,对后代的繁殖力影响显著;在相同处理浓度下,0593 较虫酰肼对甜菜夜蛾后代繁殖力的不利影响更大。因此,0593 是较虫酰肼杀虫毒力更高、对生长发育和繁殖力不利影响更大的药剂,开发应用价值很大。     

保幼激素对昆虫变态发育调控的分子机制

近年来随着保幼激素(juvenile hormone,JH)核受体Methoprene tolerant(Met)被鉴定,JH对昆虫变态发育调控的分子机制的研究取得了极大的进展。本文在介绍Met的鉴定以及分子伴侣Hsp83和核孔蛋白Nup358对Met亚细胞定位调控的基础上,重点阐述了JH-Met-Kr-h1-Br信号通路在完全变态昆虫幼虫至蛹变态过程中的作用以及JH-Met-Kr-h1-E93信号通路在不完全变态昆虫和完全变态昆虫成虫羽化过程中的作用。此外,Met与蜕皮激素(20-hydroxyecdysone,20E)受体复合物EcR/USP的结合、Tai/SRC/FISC分别与Met和EcR/USP结合形成JH功能受体和20E功能受体复合物、JH对20E下游基因E75A的诱导以及USP与JH的结合等分子间的相互作用在JH与20E的互作中所产生的影响也将逐一进行论述。本文还对JH通过膜受体激活PKC和PLC等下游信号通路而发挥生理功能的研究进展进行了概述。     

茶尺蠖蜕皮激素受体基因Eo-EcR的克隆、生物信息学分析和表达检测

【目的】蜕皮激素受体(ecdysteroid receptor,EcR)是一种超家族核受体,它能与超气门蛋白USP组成异源二聚体复合物EcR/USP,调节20-羟基蜕皮酮(20E)的生物活性,进而调控昆虫的发育、变态及繁殖等生命过程。本研究旨在克隆茶尺蠖Ectropis obliqua Prout EcR基因全长,并了解该基因的编码蛋白特征和时空表达模式。【方法】通过RT-PCR方法并结合RACE技术,克隆茶尺蠖EcR的基因全长,通过生物信息学软件和在线网站分析茶尺蠖EcR的生物学特性,通过实时荧光定量PCR(real-time quantitative PCR,qRT-PCR)技术比较茶尺蠖EcR在不同发育时期和6龄幼虫不同组织中的相对表达含量。【结果】克隆并鉴定了茶尺蠖EcR基因,将其命名为Eo-EcR(基因登录号:KP869130.1),Eo-EcR全长2 268 bp,含有1 728 bp开放阅读框,编码576个氨基酸。系统进化树和氨基酸同源性比对表明,Eo-EcR具有相对保守的进化特性,特别是与鳞翅目昆虫的保守性最高。三级结构模拟和功能结构域预测表明,Eo-EcR具有3个经典的结构模型,并以α螺旋为主,功能位点单一且为C4型锌指结构。qRT-PCR结果表明,Eo-EcR在5龄和6龄幼虫期以及成虫期表达量较高,在其他龄期表达量变化不大;同时在前胸腺表达量最高,在血淋巴表达量最低。【结论】明确了Eo-EcR的核苷酸序列及编码蛋白特征,明确了Eo-EcR的时空表达特性。该研究结果为进一步研究Eo-EcR的分子功能和基于Eo-EcR为靶标杀虫剂的研制奠定分子基础。     

Comparative toxicity of three ecdysone agonist insecticides against the Mediterranean flour moth

The Mediterranean flour moth, Ephestia Kuehniella Zeller (Lepidoptera: Pyralidae), is an important pest in stored products worldwide, and is one of the major pests in flour mills in Algeria. Because environmental consideration, alternative approaches to neurotoxic insecticides, as well as safe, effective, and sound integrated pest management strategies are developed pest control agents such as the insect growth regulator (IGRs). Among these IGRs, the bisacylhydrazine derivatives are nonsteroidal ecdysterold agonists that mimic the action of moulting hormones and induce a precocious and incomplete moult in several insect orders. In topical bioassays using the pupae of E. kuehniella, three ecdysteroid agonists: RH-5849, the first bisaclhydrazine ecdysone agonist and two analogs, RH-5992 (tebufenozide) and RH-0345 (halofenozide), were evaluated on the reproduction under laboratory conditions. In a first series of experiments, the efficacy of these compounds was tested. These compounds exhibited insecticidal activity and the duration of pupal development was reduced with a dose-response relationship. Among the three tested compounds, tebufenozide (LD50 = 0.005 microg) appeared the most potent ecdysteroid agonist against E. kuehniella (RH-5849: LD50 = 0.05 microg and RH-0345: LD50 = 5.10 microg). In a second series of experiments, the effects of the ecdysone agonists (LD50) were investigated on the reproduction. Data showed that the three compounds affected growth of ovaries as evidenced by morphometric measurements of the ovaries from newly emerged adult females. In addition, the thickness of the chorion from basal oocytes was reduced only by RH-5992 and RH-0345. However, electron microscopic observations revealed that the three compounds had no significant effect on the fine structure of chorion. Finally, measurements of ovarian ecdysteroids' production by an enzyme immunoassay showed an increase in the hormonal amounts recorded in treated series compared to control series.     

Towards Coleoptera-specific high-throughput screening systems for compounds with ecdysone activity: development of EcR reporter assays using weevil (Anthonomus grandis)-derived cell lines and in silico analysis of ligand binding to A. grandis EcR ligand-binding pocket

Molting in insects is regulated by ecdysteroids and juvenile hormones. Several synthetic non-steroidal ecdysone agonists are on the market as insecticides. These ecdysone agonists are dibenzoylhydrazine (DBH) analogue compounds that manifest their toxicity via interaction with the ecdysone receptor (EcR). Of the four commercial available ecdysone agonists, three (tebufenozide, methoxyfenozide and chromafenozide) are highly lepidopteran specific, one (halofenozide) is used to control coleopteran and lepidopteran insects in turf and ornamentals. However, compared to the very high binding affinity of these DBH analogues to lepidopteran EcRs, halofenozide has a low binding affinity for coleopteran EcRs. For the discovery of ecdysone agonists that target non-lepidopteran insect groups, efficient screening systems that are based on the activation of the EcR are needed. We report here the development and evaluation of two coleopteran-specific reporter-based screening systems to discover and evaluate ecdysone agonists. The screening systems are based on the cell lines BRL-AG-3A and BRL-AG-3C that are derived from the weevil Anthonomus grandis, which can be efficiently transduced with an EcR reporter cassette for evaluation of induction of reporter activity by ecdysone agonists. We also cloned the almost full length coding sequence of EcR expressed in the cell line BRL-AG-3C and used it to make an initial in silico 3D-model of its ligand-binding pocket docked with ponasterone A and tebufenozide.     

Effect of ecdysone agonists on vitellogenesis and the expression of EcR and USP in codling moth (Cydia pomonella)

The effects of tebufenozide and methoxyfenozide on vitellogenin (Vg) synthesis/release in the fat body, translocation in hemolymph, uptake by the ovary, and the expression of the ecdysone receptor (EcR) and its heterodimer partner, ultraspiracle protein (USP) in fat body, were investigated in Cydia pomonella. The results indicated that both ecdysone agonists significantly increased the Vg level in the adult hemolymph when the moths were exposed to agonist-treated surfaces. However, these agonists did not affect Vg release from the fat body nor Vg deposition in the first batch oocytes. Western blot analysis revealed that the expression of EcR and USP was significantly increased in tebufenozide- and methoxyfenozide-treated samples compared to the control, suggesting that ecdysone agonists regulated the Vg synthesis via the EcR and USP proteins complex.     

Significance of absorption, oxidation, and binding to toxicity of four ecdysone agonists in multi-resistant cotton leafworm

Treatment of last-instar larvae of multi-resistant cotton leafworm Spodoptera littoralis with four dibenzoylhydrazines, methoxyfenozide (RH-2485), tebufenozide (RH-5992), halofenozide (RH-0345), and RH-5849, resulted in premature molting leading to death. Methoxyfenozide was the most toxic followed by tebufenozide, halofenozide, and RH-5849. To explain differences in toxicity, especially between multi-resistant and laboratory strains, absorption in the body tissues and oxidative metabolism were tested with 14C-labeled ecdysone agonist and a Lineweaver-Burk assay, respectively. Then to address different compound potencies in multi-resistant strains, the potency of the four ecdysone agonists was measured based on their ability to mimic the natural insect molting hormone, 20-hydroxyecdysone (20E) by inducing evagination in isolated imaginal wing discs. Using monoclonal antibody 9B9, the presence of ecdysteroid receptors in imaginal discs in vitro was confirmed. In parallel, Scatchard plot analysis with whole imaginal wing discs cultured with different concentrations of 3H-labeled ponasterone A indicated no significant difference in affinity and in number of target sites for binding between multi-resistant and susceptible laboratory strains. The four compounds tested caused the effect as agonists of 20E in vitro, and typically the order of their toxicities (LC50s) corresponded with that for evagination-induction with whole imaginal discs.     

Action of 24-epibrassinolide on a cell line of the beet armyworm, Spodoptera exigua

The Spodoptera exigua cell line Se4 is sensitive for ecdysteroid activity stimulated by the insect molting hormone, 20-hydroxyecdysone (20E), showing a cease in cell proliferation (with 50% inhibition around 1 microM) and characteristic cell morphology changes with aggregation and formation of long filamentous cytoplasmic extensions. The bisacylhydrazine tebufenozide also triggered such typical cellular effects in Se4, and in addition, it showed an affinity for binding in competition with 3H-ponasterone A (PoA) that was similar to 20E (with 50% competition around 1 microM), confirming that such non-ecdysteroids display an ecdysteroid agonist activity. In contrast, when Se4 cells were incubated with the native plant hormone 24-epibrassinolide (24BR), none of the effects triggered by 20E were observed. Hence, a competition binding experiment with 3H-PoA demonstrated no affinity of 24BR for binding to the ecdysteroid receptor in the Se4 cell line. In another series of experiments, the Se4 cell line was tested in sensitivity response to increased acetylcholinesterase (AchE) activity after treatment with ecdysteroid active compounds. The AchE activity measured in the cell line is discussed in relation to inhibition by eserine. The obtained results suggest that 24BR exerted no ecdysteroid activity.     

Ecdysteroid levels throughout the life cycle of the desert locust, Schistocerca gregaria

Moulting hormone levels for all stages of the life cycle of the desert locust, Schistocerca gregaria, have been determined using gas chromatography with electron capture detection of the trimethylsilylated hormones. During larval development, the major hormone detected is 20-hydroxyecdysone with smaller quantities of ecdysone present. In mature adult females the major ecdysteroid observed is a polar conjugate of ecdysone, with smaller quantities of conjugated 20-hydroxyecdysone also present. During embryonic development the pattern changes from a high proportion of conjugated ecdysone in the early stages to give more free hormone and a higher proportion of 20-hydroxyecdysone in later stages. The highest titre of 20-hydroxyecdysone found in this insect is during the 5th larval instar. Maximal levels of ecdysteroid per insect are found in mature females just before oviposition, while the highest level of ecdysteroid per g of tissue is found in the eggs.     

Distribution and metabolism of exogenous ecdysteroids in the egyptian cotton leafworm Spodoptera littoralis (lepidoptera: noctuidae)

After ingestion of various amounts of either [³H]ecdysone or [³H]20-hydroxyecdysone (0.8 ng to 10 μg) by sixth instar larvae of the Egyptian cotton leafworm Spodoptera littoralis, apolar metabolites are rapidly detected in the gut and frass. Hydrolysis of the apolar products with Helix hydrolases releases solely [³H]ecdysone or [³H]20-hydroxyecdysone, respectively. This, coupled with the formation of chemical derivatives (acetonide and acetate) which cochromatograph with authentic reference compounds on hptlc and hplc demonstrates that these apolar metabolites consist of ecdysone or 20-hydroxyecdysone esterified at C-22 with common long-chain fatty acids. The major fatty acids have been identified by RP-hplc and their contribution to the mixture determined. In contrast, [³H]ecdysone injected into the haemolymph of S. littoralis is metabolized to yield 20-hydroxyecdysone, ecdysonoic acid, and 20-hydroxyecdysonoic acid. Thus, two different pathways exist for the metabolism of ecdysteroids in this species. In addition to an essentially polar pathway operating on injected and endogenous ecdysteroids, exogenous ecdysteroids entering the gut of S. littoralis are detoxified, yielding apolar ecdysteroid 22-fatty acyl esters which are rapidly excreted. The significance of these results in relation to the effects of ingested ecdysteroids on S. littoralis is discussed. Arch. Insect Biochem. Physiol. 34:329–346, 1997. © 1997 Wiley-Liss, Inc.     

Creation of ecdysone receptor chimeras in plants for controlled regulation of gene expression

Transformation with a chimeric receptor containing the glucocorticoid transactivation and DNA-binding domains fused to an ecdysteroid receptor ligand-binding domain permits ecdysone agonist-inducible gene expression in monocotyledonous plant cells. The inducible system is based on the specific activation of a chimeric receptor containing the ligand-binding domain of the Heliothis virescens ecdysteroid receptor and the inducer RH5992 (a 20-hydroxyecdysone agonist). RH5992 is an non-steroidal agrochemical with a high specificity for lepidopteran ecdysone receptors. Addition of RH5992 to transformed cells results in high levels of inducible expression in a ligand-specific manner, particularly when the effector receptor is coupled to the strong transactivator VP16. A chimeric construct containing the Drosophila ecdysone ligand-binding domain failed to activate reporter gene activity with RH5992, while activation was observed in the presence of muristeroneA. The system described provides the basis for an inducible gene expression system that is compatible with agricultural use. Received: 24 September 1998 / Accepted: 15 January 1999