桔小实蝇蜕皮激素合成和信号通路基因调控与功能研究
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摘要
桔小实蝇Bactrocera dorsalis (Hendel)隶属于双翅目Diptera,实蝇科Tephritidae,是一种在世界范围内广泛分布的重要农业害虫。桔小实蝇成虫产卵于果皮内,幼虫孵化后在果实内取食危害,严重降低果实经济和食用价值；加之其成虫寄主范围广、适应性和繁殖力以及扩散能力强等特点,该虫被许多国家列为检疫对象,成为国际贸易的技术壁垒。与其他昆虫一样,桔小实蝇不能依赖自身物质从头合成蜕皮激素,只能是将其取食获得的植物甾醇或动物胆固醇进行一系列相应的代谢而产生。负责该过程的是一类名为‘'Halloween gene"的P450多功能氧化酶,将植物甾醇最终氧化成为最具生物活性的20-羟基蜕皮酮(20E),调控昆虫胚胎发育、幼虫生长与变态、蛹期形态建成以及成虫交配繁殖等整个过程。合成的20E可诱导蜕皮激素受体EcR表达,当EcR达到一定阈值后,与其配体USP形成功能性的EcR/USP异二聚体。随后,再与20E进行结合,形成20E-EcR/USP复合体,起始20E信号传递,并介导产生一系列早期和晚期级联反应,最终影响细胞的增殖、分化,甚至程序性死亡。随着RNAi技术的出现与成熟,基因功能的研究进入一个崭新的阶段。借助该技术,可以重新发现基因新功能、探索未知基因功能,是生物反应、药物筛选、疾病治疗、害虫防治等研究的有力手段,极大地加快了分子生物学研究步伐,具有广阔的应用前景。
本学位论文以柑桔重要害虫桔小实蝇为研究对象,利用RT-PCR和RACE技术,克隆获得桔小实蝇蜕皮激素合成和信号通路基因全长序列,对其特征序列进行注释并确定其系统发育关系；在此基础上运用qPCR技术分析这些基因在桔小实蝇胚后发育各阶段和幼虫不同组织中的表达及分布情况；同时,结合生测试验,明确幼虫在饥饿胁迫条件下的生物学变化和上述基因转录水平情况,明确桔小实蝇的应激响应机制,为其综合防治提供依据。昆虫生长发育是受激素严格控制的过程,在农业生产中常用激素类似物质来干扰害虫的正常生长,从而达到控害的目的。因此,本研究还利用微量注射技术,研究外源20E对桔小实蝇幼虫发育、变态及蜕皮激素合成和信号通路基因表达的影响。最后,利用RNAi技术明确桔小实蝇蜕皮激素受体BdEcR-B1在幼虫阶段的20E信号传导中的作用。加深桔小实蝇变态发育的分子调节和作用机制的研究,尤其是对蜕皮激素合成和信号传导通路对其发育变态的分子调控模式,及其在逆境胁迫条件下的应激响应机制,将有助于明确各基因在通路中的功能及其调节方式,为转基因技术在害虫控制中的应用提供潜在作用靶标,同时为新型农药开发提供理论依据。本研究主要研究结果如下：
1桔小实蝇蜕皮激素合成与信号通路基因克隆与序列分析
基于对桔小实蝇转录组数据分析的基础上,利用RT-PCR和RACE技术,克隆获得了桔小实蝇蜕皮激素合成和信号通路上7个基因的cDNA全长序列,并完成在GenBank登录,其基因名称和登录号分别为BdCyp302α1(JQ027284)、 BdCyp315α1(KC515377)、BdCyp314α1(JQ229645)、BdEcR-B1(JQ034623)、 BdE75(KC515378)、BdL63(KC515379)和BdTaiman (JQ266268)。利用分子生物学软件对上述基因进行分析,明确其开放阅读框ORF、氨基酸序列、理化性质、跨膜结构域等,并对其保守结构域进行了鉴定和详细注释。分析发现,桔小实蝇蜕皮激素合成通路基因BdCyp302α1、BdCyp315α1和BdCyp314α1均属于"Halloween"基因家族,具有细胞色素氧化酶P450所特有签名序列：Helix-I、 Helix-K、底物识别性位点和血红蛋白结合位点等结构。桔小实蝇的BdCyp302α1和BdCyp314α1属于线粒体型,而BdCyp315α1属于微粒体细胞色素氧化酶。在蜕皮激素信号通路基因的研究中发现,桔小实蝇BdEcR-B1具有典型核受体保守结构域和微结构域,如DNA结合域和配体结构域等结构,双翅目昆虫EcR-B1亚型所特有的K/RRRW、S-rich、DL-rich、EESST/SEVV/TSS等特征基序。与BdEcR-B1相同,BdE75同属于核受体基因家族,具有其典型的配体结合域以及DNA结合域,其内含量2个C4锌指结构,在蜕皮激素信号通路上起初级响应元件的作用。BdL63序列N端具有cyclin-dependent kinase结构。BdTaiman属于p160核受体共激活子家族,具有basic helix-loop-helix (bHLH)结构域及乙酰化典型结构基序LxxLL。
2桔小实蝇蜕皮激素合成与信号通路基因的表达模式分析
2.1桔小实蝇蜕皮激素合成与信号通路基因在不同发育阶段的表达模式
在成功建立桔小实蝇蜕皮激素合成与信号通路基因的qPCR反应体系的基础上,以a-Tubulin为内参基因,分别对桔小实蝇蜕皮激素合成通路(BdNvd、 BdCyp306α1、BdCyp302α1、BdCyp315α1和BdCyp314α1)和信号通路(BdEcR-B1、 BdUSP、BdE75、BdL63、BdTaiman)基因在其不同发育阶段(幼虫、蛹和雌成虫),共25个时间点的表达情况进行了分析。
桔小实蝇蜕皮激素合成通路基因在不同日龄幼虫中的表达模式较为相似,BdNvd、BdCyp302α1、BdCyp314α1基因的最高表达量均出现在末龄幼虫阶段,与即将出现的化蛹行为有关。在蛹期,与BdNvd在蛹末期高表达不同,BdCyp306α1、 BdCyp302α1和BdCyp315α1基因相对最高表达量出现在化蛹初期,并随着蛹日龄的增加而显著地降低；BdCyp314α1则在蛹期呈现3个表达高峰,可能与20E介导的组织重建过程相关。在成虫期,除BdCyp314α1外,桔小实蝇蜕皮激素合成通路基因的最低表达量均出现在羽化初期,在第7日龄中出现显著性升高,并随即下降后再次上升,并在第19日龄成虫中达到相对表达量的最大值,表明蜕皮激素合成通路基因的表达与其性成熟和产卵行为有关。
就蜕皮激素信号通路基因而言,BdEcR-B1、BdUSP、BdL63和BdTaiman的表达量在末龄幼虫阶段均出现上升,暗示这些基因可能与蜕皮激素介导的变态反应有关。BdE75在蛹中期特异性高表达,可能在组织重建中具有重要作用。其余基因在蛹中后期表达模式比较相似,呈现逐渐下降的趋势,暗示由20E调控的组织重建过程也趋于完成。在成虫期,BdEcR-B1和BdE75基因的相对表达量随日龄的增加而出现上升；BdUSP和BdL63基因的相对表达量在前16日中保持相对稳定,在第19日龄出现明显的上升；而BdTaiman基因的相对最高表达量出现在雌虫羽化的第1天,并在随后的阶段保持相对稳定,推测上述基因在雌虫卵巢成熟过程中起不同作用。
2.2桔小实蝇蜕皮激素合成与信号通路基因在不同组织的表达模式
同样以a-Tubulin为内参基因,对上述基因在桔小实蝇5日龄幼虫前胸腺(复合体)、脂肪体、中肠、马氏管、表皮和气管的mRNA的表达水平进行了评价。结果发现,前胸腺仍为蜕皮激素早期合成的主要场所。BdNvd、BdCyp306α1和BdCyp315α1基因在前胸腺中特异性高表达,极显著高于脂肪体、中肠、马氏管、表皮和气管,而这些组织内的表达量无显著性差异。BdCyp302α1基因在脂肪体、前胸腺、马氏管／表皮、中肠／气管中的表达量逐步降低。而BdCyp314α1基因在中肠、马氏管、脂肪体中的相对表达量依次降低,但均显著性高于表皮、气管和前胸腺。说明桔小实蝇不同组织均参与蜕皮激素的合成,前胸腺、脂肪体和中肠是20E合成的主要场所：此外,马氏管除主要负责排泄外,也参与了蜕皮激素的合成。
在桔小实蝇蜕皮激素信号通路基因的研究中发现,BdEcR-B1和BdE75的表达模式较为相似,在脂肪体的表达量极显著高于其余5个组织,且在5个组织内无显著性差异；其中,BdE75在脂肪体内特异性高表达,其表达量是表皮的122倍。BdUSP在前胸腺表达量与脂肪体无显著性差异,但均极显著高于其他4个组织。而BdL63的表达量在脂肪体、前胸腺和马氏管中依次降低,而在马氏管、中肠和表皮中无显著性差异。BdTaiman基因在马氏管中的表达量显著性高于表皮,但与前胸腺、脂肪体和中肠差异不显著。综上,蜕皮激素信号通路基因主要集中在脂肪体内表达,由此推测,这可能与脂肪体营养储存和能量代谢功能相关。
3饥饿对桔小实蝇幼虫发育和蜕皮激素合成与信号通路基因的影响
3.1饥饿对桔小实蝇幼虫发育的影响
对桔小实蝇5日龄幼虫进行不同时长饥饿处理后发现,饥饿有效缩短幼虫历期,造成提前化蛹,且蛹体较小的现象,但并未引起幼虫存活率的降低,说明桔小实蝇在末龄幼虫的第1天,已经达到其化蛹所需最低体重阈值；在食物不足的情况下,通过提前变态成小个体蛹,来避免外界不利环境对自身的影响,维持种群延续。
3.2桔小实蝇蜕皮激素合成信号通路基因在饥饿胁迫下的表达模式
利用实时定量荧光PCR检测发现,其蜕皮激素合成通路基因表达水平在饥饿处理后出现明显的变化。BdNvd、BdCyp302α1和BdCyp314α1基因的相对表达量在饥饿胁迫6h后即出现显著性上调,其中BdCyp302α1和BdCyp314α1基因在24h达到极显著上调水平：而饥饿后48h后,BdNvd、BdCyp302α1和BdCyp314α1的表达水平出现显著性降低。对信号通路基因的研究发现,除BdL63外,其余各基因表达均出现显著性上调。由此推测,桔小实蝇幼虫在食物缺乏的状况下,可能通过提高蜕皮激素合成和信号通路基因的转录水平使体内蜕皮激素含量升高,信号传导加强,导致其幼虫提前化蛹,从而避免不良环境的胁迫。
4外源20E对桔小实蝇幼虫发育和蜕皮激素合成与信号通路基因的影响
4.1外源20E对桔小实蝇幼虫发育的影响
利用微量注射法,观察不同剂量20E处理对桔小实蝇幼虫发育的影响。结果发现,注射激素后,幼虫化蛹时间提前。同时,化蛹过程出现异常,并出现头部缢缩、黑化以及幼虫形态蛹的现象,且畸形的蛹宽和蛹长出现变化。畸形率和蛹死亡率显著上升。说明外源激素干扰了桔小实蝇幼虫正常生长发育和变态过程。
4.2外源20E对桔小实蝇蜕皮激素合成与信号通路基因的影响
经0.1μg/头剂量处理后,桔小实蝇蜕皮激素合成通路基因BdNvd、 BdCyp306α1、BdCyp302α1和BdCyp315α1均表现出相似的诱导反应,其相对表达量出现极显著上调,而BdCyp314α1的表达却被外源激素所抑制。桔小实蝇蜕皮激素信号通路基因的表达也表现出类似的诱导效应。除BdEcR-B1仅在处理1h后出现极显著的下调外,其它基因较对照而言,均出现极显著的上调。
经0.1和1.0μg/头外源20E处理后,BdNvd、BdCyp306α1和BdCyp315α1的表达量均极显著高于对照组；BdCyp302α1的表达量仅在0.1μg/头的剂量处理下极显著性高于对照组；而BdCyp314α1的表达量在0.5和1.0μg/头处理组的表达量均显著性低于对照。此外,BdEcR-B1和BdUSP均出现极显著上调。BdE75和BdL63对20E处理的反应较为一致,均在0.1μg和1.0μg/头处理后出现极显著性的上调。但0.5μg/头处理对蜕皮激素合成和信号通路基因的表达均表现出不同程度的抑制作用,该剂量可能是桔小实蝇体内某些生理反应的一个阈值,影响其生长发育等重要过程。
5桔小实蝇BdEcR-B1基因的功能研究
通过体外转录法获得高质量dsRNA,借助微量注射技术将外源dsRNA导入桔小实蝇5日龄幼虫体内。qPCR分析结果表明,BdEcR-B1基因表达量在注射72h后出现明显的下调,其表达量仅为对照组的53.4%。对桔小实蝇蜕皮激素信号通路基因的相对表达量进行分析发现,在靶基因沉默后,BdUSP和BdTaiman基因相对表达量出现了显著性下调,其中BdTaiman基因相对表达量在注射24-72h后出现极显著的下调,仅为对照组的9.7-12.5%。而BdE75基因和BdL63基因的转录并未受影响,出现2倍左右的上调。说明BdEcR-B1在蜕皮激素信号传导中直接调控其配体和共激活子的表达。
综上所述,本研究通过高通量测序、RT-PCR和RACE技术,分离克隆获得桔小实蝇蜕皮激素合成和信号通路基因cDNA全长序列,借助qPCR手段分析其在不同发育阶段、不同组织、饥饿胁迫以及激素处理条件下的表达模式,明确其调控机制和表达规律。同时,利用生测技术,研究饥饿和外源20E对桔小实蝇幼虫发育的影响。在此基础上,进一步利用RNAi技术,验证BdEcR-B1在蜕皮激素信号级联反应中的中心地位。研究结果有助于阐明蜕皮激素对昆虫发育和变态的调控机制,加深蜕皮激素合成与信号基因功能的认识,并为以上述通路基因为靶标的害虫防治方法提供理论依据和实践价值。
The oriental fruit fly, Bactrocera dorsalis (Hendel)(Diptera, Tephritidae) is a world-wide devastating agricultural pest. The adults lay eggs underneath the exocarp, and the larvae hatched and fed on the fruit, causing direct fruit damage and economic loss. Moreover, due to the wide host ranges, high adaptive capacity, fecundity and dispersal ability, B. dorsalis is documented on the quarantine target lists in many countries and treated as an export barrier in the international trade. Insects lack the de novo pathway of synthesizing20-hydroecdysone (20E), instead,20E is synthesized from dietary cholesterols or phytosterols. The mechanism involved in ecdysone biosynthesis is mediated by several P450enzymes, encoded by the Halloween gene family.20E, the most active form of ecdysteroid, acts as a critical hormone signal to coordinate insect embryonic development, larval growth and metamorphosis, pupal remodeling, courtship and reproduction precisely during the whole biological processes. The ecdysone receptor (EcR) could be induced by20E, when the expression level of EcR exceeds the threshold, EcR was assembled with ultraspiracle (USP) into a functional heterodimer receptor. After being assembled, the20E-EcR/USP complex sets off a series of early and late cascade responses, regulating cell proliferation, differentiation, or even cell programmed death. With RNAi technology's emerging and maturing, the gene functional study stepped into a new era. It is a very powerful tool in biological functional study, pharmacological screening, genetic therapy, pest management et al, which has greatly advanced the research of molecular biology and may have a prosperous future.
This dissertation focused on the ecdysone synthesis and signaling pathway of B. dorsalis, an economically important citrus pest. Firstly, the aforementioned genes involved in these two pathways were cloned, identified and detailed annotated using RT-PCR and RACE technologies as well as bioinformatics software. Secondly, the expression profiles of these genes were analyzed in different developmental stages and tissues. Bioassay and qPCR were further applied to evalute the effect of starvation in biological and molecular responses under nutrient stress. Insect growth regulators were often used in pest control, in the purpose of unbalancing the endocrinal signal of insects, causing its death. The impacts on the growth, metamorphosis of the larvae and expression profiles of aforementioned genes were tested after injected with exogenous20E. Finally, the function of BdEcR-Bl in ecdysone signaling cascade was interpreted by RNAi. The study on molecular regulation mechnism of metamorphsis and development, especially the ecdysone synthesis and signaling pathways, could deepen the understanding of function and regulation mechanism of the genes in the fields, providing potentional tagrets and theoretical basis in transgenic food applying and new insectcides screening in pest control. The main results are as follows:
1Molecular cloning and characterization of ecdysone synthesis and signaling pathway genes of B. dorsalis
Based on the high-throughput transctiptome sequencing of B. dorsalis,7novel genes were isolated with RT-PCR and RACE technologies, and deposited in GenBank with the following names and accession numbers:BdCyp302α1(JQ027284), BdCyp315α1(KC515377), BdCyp314α1(JQ229645), BdEcR-B1(JQ034623), BdE75(KC515378), BdL63(KC515379) and BdTaiman (JQ266268). The open reading frames (ORF), pupative amino acids sequences, physico-chemical properties, transmembrane regions and conserved domains were predicted and annotated in detail. The results indicated that BdCyp302α1, BdCyp315α1and BdCyp314α1in ecdysone synthesis pathway, were the members of Halloween gene family, and harbored the typical signature motifs of P450enzymes, such as Helix-I, Helix-K, substrate recognition site (SRS) and heme-binding domains. BdCyp302α1and BdCyp314α1were classified into mitochondrial, while BdCyp315α1was classified into microsomal type of P450gene. BdEcR-B1contained several typical nuclear receptor structures, such as DNA-binding domain (DBD), ligand-binding domain (LBD), and EcR-B1isoform-specific motifs like K/RRRW, S-rich, DL-rich, EESST/SEVV/TSS, which especially contained in Diptera. BdE75also belonged to nuclear receptor family with DBD, LBD and two zinc finger motifs, acting as an early response element in ecdysone signaling cascade. At the N-terminus, BdL63possessed a cyclin-dependent kinase domain. Besides, BdTaiman was identified to be a member of p160family of nuclear receptor coactivator, harboring basic helix-loop-helix (bHLH) and classical acetylation motif LxxLL.
2Expression patterns of ecdysone synthesis and signaling pathway genes of B. dorsalis
2.1Expression patterns of ecdysone synthesis and signaling pathway genes in different stages
Total RNA of different developmental stages was extracted from the larvae (1-day-old to8-day-old with one-day interval), pupae (1day-old to10day-old with one-day interval) and female adults (1day-old to19day-old with three-day interval) of B. dorsalis. On the one hand, the expression profiles of5genes functioning in ecdysone synthesis pathway, BdNvd, BdCyp306α1, BdCyp302al, BdCyp315al, BdCyp314α1and5signaling pathway genes, BdEcR-B1, BdUSP, BdE75, BdL63and BdTaiman were estimated in those samples by qPCR, using a-Tubulin as an internal control. The results showed that, during the larval stages, BdNvd, BdCyp302al and BdCyp314al shared the same profile patterns, with the highest expression levels occurred in the mature larvae, suggesting they might participate in the following pupation course. During the pupal stages, BdCyp306al, BdCyp302α1and BdCyp315al were highly expressed at the very beginning of this stage, and declined significantly with the increase of time, different from the expression pattern of BdNvd. The relative expression of BdCyp314α1was demonstrated with3peaks during the pupal stage, implying it might play an important role in tissue remodeling. In newly emerged adults, the relative expressions levels of BdNvd, BdCyp306α1, BdCyp302α1and BdCyp315al were the lowest, and significantly rised at the seventh day, then decreased and rised gradually, reached the maxium level at the19-day-old female, indicating they could involve in sex maturation and oviposition.
On the other hand, ecdysone signaling pathway genes BdEcR-B1, BdUSP, BdL63and BdTaiman were highly transcribed during the late larval stage, involving in coordinating the ecdysone signal with metamorphosis at this stage. BdE75was highly expressed in the middle pupal stage, implying its key role in tissue remodeling. The other signaling pathway genes shared the similar expression patterns during pupal stage, which decreased gradually during the middle-late couse, suggesting the procedure of adult morphogenesis tend to be complete under the regulation of20E. After eclosion, the expression levels of BdEcR-B1and BdE75gradually rised; BdUSP and BdL63significantly rised at the19th day. Besides, BdTaiman highly expressed in the1-day-old females, and remained stable in the following fifteen days, implying their different roles in ovary maturation.
2.2Expression patterns of ecdysone synthesis and signaling pathway genes in different tissues
The relative expression levels of ecdysone synthesis and signaling pathway genes were evaluated in six tissues including prothoracic glands (mixture), fatbody, midgut, Malpighian tubules, integument and trachea, which were dissected from the5-day-old larvae of B. dorsalis, with a-Tubulin as a reference gene. The results revealed that the prothoracic glands remain to be the main site which involved in early step of20E synthesis. BdNvd, BdCyp306α1and BdCyp315α1very highly expressed in prothoracic glands, and their expression levels showed no significant difference in fatbody, midgut, Malpighian tubules, integument and trachea. The expression profiles of BdCyp302α1were successively decrease in fatbody, prothoracic glands, Malpighian tubules/integument and midgut/integument. Furthermore, the expression levels of BdCyp314α1were highly expressed in midgut, Malpighian tubules and fatbody. The results indicated that, although many tissues participated in ecdysone synthesis, prothoracic glands, fatbody and midgut remained to be the main sites for the process. In addition to excretion, Malpighian tubules were also in charge of ecdysone biosynthesis.
The expression levels of signaling pathway genes BdEcR-B1and BdE75shared the same tissue distribution pattern, whose expression levels in the fatbody were relatively higher than those in the other five tissues. In additin, BdE75was exclusively highly expression in fatbody. The mRNA expression levels of BdUSP showed no significant differences in prothoracic glands and midgut, but sigfinficantly higher than those in midgut, Malpighian tubules, integument and trachea. The expression profiles of BdL63were successively decrease in fatbody, prothoracic glands and Malpighian tubules, however, there were no difference among Malpighian tubules, midgut and integument. BdTaiman showed a higher expression level in the Malpighian tubules than in integument, but no significant difference between Malpighian tubules, fatbody and midgut. The results showed that the20E signaling pathway genes predominately expressed in the fatbody, indicating those genes might be involved in nutrients storage and energy utilization of this certain tissue.
3The effects of starvation on larvae development and expression profiles of ecdysone synthesis and signaling pathway genes of B. dorsalis
3.1The effects of starvation on the larvae development of B. dorsalis
Different food deprivation time were applied to the5-day-old larvae to illustrate the stress response of B. dorsalis. The data showed that starvation could shorten the larval duration, causing prepupation, but no side effect on survival rate, indicated the body weight of the5-day-old larvae, the first day of the third instar, exceed the thredhold value of pupation. Moreover, when food was insufficient, the larvae metamorphosis precociously, avoiding harmful environmental stress.
3.2Expression patterns of ecdysone synthesis and signaling pathway genes under starvation stress
In addition to bioassay, qPCR was applied to illustrate the stress response of ecdysone synthesis and signaling pathway genes under starvation. The expressions levels of BdNvd, BdCyp302al and BdCyp314al were signicantly elevated at6h post food deprivation, and BdCyp302al and BdCyp314al were very highly elevated at24h. At48h, the expressions of BdCyp302α1and BdCyp314α1were significantly lower than that of the control. Except for BdL63, ecdysone signaling pathway genes were all significantly up-regulated. These results suggested that nutritional deprivation could cause a rapid rise of20E titer and strengthened signals by up-regulating the expression levels of ecdysone synthesis and signaling pathway genes, which would ultimately lead to pupate precociously, avoiding the detrimental effects of the adverse environmental stress.
4The effects of exogenous20E on larvae development and expression profiles of ecdysone synthesis and signaling pathway genes of B. dorsalis
4.1The effects of exogenous20E on the larvae development of B. dorsalis
This study has been carried out to investigate the effects of exogenous20E on the5-day-old larvae of B. dorsalis by injecting different doses of20E. The results showed that20E caused precocious pupation, extraordinary phenomena, including abnormal pupation in artificial diet, shrunken, melanized and larval-formed pupae. The length, width and malformation rate were significantly different between the control and treatment. The results also showed that there may be a positive correlation between mortality and the dose of20E, which indicating that the exogenous20E had a remarkable negative effect on the growth and metamorphosis of B. dorsalis.
4.2The effects of exogenous20E on expression profiles of ecdysone synthesis and signaling pathway genes of B. dorsalis
The expressions of ecdysone synthesis pathway genes BdNvd,BdCyp306α1, BdCyp302al and BdCyp315α1showed a similar induced effect after injection with0.1μg/insect of exogenous20E, which were increased significantly. However, the expression of BdCyp314α1was depressed by exogenous20E. The expression profiles of ecdysone signaling pathway genes showed the same induced effects, which were highly induced, except for BdEcR-B1at1h after injection.
After injection with0.1and1.0μg/insect of exogenous20E, the expression levels of BdNvd, BdCyp306α1and BdCyp315α1were significantly elevated compared to the control, but BdCyp302α1was highly upregulated by0.1Lg20E. However, the expression of BdCyp314α1was significantly down-regulated after injection with0.5and1.0μg/insect of20E. In addition, BdEcR-B1and BdUSP were up-regulated. The expressions of BdE75and BdL63were increased by injection with0.1and1.0μg/insect20E. However, the0.5μg/insect of20E suppressed the expressions of both ecdysone synthesis and signaling pathway genes in varying dgree, indicating this could be a threshold dose in some important physiological reaction, intefering the development of B. dorsalis.
5Functional study of BdEcR-B1of B. dorsalis
High quality of dsRNA was synthesized by in vitro transcription, and delivered into B. dorsalis by microinjection. Quantitative PCR showed that, the expression level of BdEcR-B1was reduced significantly compared to dsGFP treatement after injected for72h, and so did in BdUSP and BdTaiman. Especially, the expression of BdTaiman was depressed to a very low level, down-regulated87.5-90.3%compared to the control. However, BdE75and BdL63showed2-fold up-regulated after injection. The results indicated that BdEcR-Bl play an important role in controlling the expression levels of BdUSP and BdTaiman, which worked as its heterodimer partner and coactivator in ecdysone signaling pathway.
Taken together, the full-length of ecdysone synthesis and signaling pathway genes were obtained by high-throughput sequencing, RT-PCR and RACE methods. Quantitative PCR was applied to illustrate the expression patterns of these genes in different stages and tissues, and under starvation stress and hormone interference conditions. Meanwhile, bioassay strategy was utilized to illuminate the response to starvation and exogenous20E on larvae development. Further, based on RNAi, the central role of BdEcR-B1in ecdysone signal transduction was confirmed in B. dorsalis. All these results will not only elucidate the molecular mechanism of ecdysone regulation on growth and metamorphosis, deepen our understanding the functions of the genes in ecdysone synthesis and signaling pathway, but also provide theoretical basis for EcR-targeting pest control.
本学位论文以柑桔重要害虫桔小实蝇为研究对象,利用RT-PCR和RACE技术,克隆获得桔小实蝇蜕皮激素合成和信号通路基因全长序列,对其特征序列进行注释并确定其系统发育关系；在此基础上运用qPCR技术分析这些基因在桔小实蝇胚后发育各阶段和幼虫不同组织中的表达及分布情况；同时,结合生测试验,明确幼虫在饥饿胁迫条件下的生物学变化和上述基因转录水平情况,明确桔小实蝇的应激响应机制,为其综合防治提供依据。昆虫生长发育是受激素严格控制的过程,在农业生产中常用激素类似物质来干扰害虫的正常生长,从而达到控害的目的。因此,本研究还利用微量注射技术,研究外源20E对桔小实蝇幼虫发育、变态及蜕皮激素合成和信号通路基因表达的影响。最后,利用RNAi技术明确桔小实蝇蜕皮激素受体BdEcR-B1在幼虫阶段的20E信号传导中的作用。加深桔小实蝇变态发育的分子调节和作用机制的研究,尤其是对蜕皮激素合成和信号传导通路对其发育变态的分子调控模式,及其在逆境胁迫条件下的应激响应机制,将有助于明确各基因在通路中的功能及其调节方式,为转基因技术在害虫控制中的应用提供潜在作用靶标,同时为新型农药开发提供理论依据。本研究主要研究结果如下：
1桔小实蝇蜕皮激素合成与信号通路基因克隆与序列分析
基于对桔小实蝇转录组数据分析的基础上,利用RT-PCR和RACE技术,克隆获得了桔小实蝇蜕皮激素合成和信号通路上7个基因的cDNA全长序列,并完成在GenBank登录,其基因名称和登录号分别为BdCyp302α1(JQ027284)、 BdCyp315α1(KC515377)、BdCyp314α1(JQ229645)、BdEcR-B1(JQ034623)、 BdE75(KC515378)、BdL63(KC515379)和BdTaiman (JQ266268)。利用分子生物学软件对上述基因进行分析,明确其开放阅读框ORF、氨基酸序列、理化性质、跨膜结构域等,并对其保守结构域进行了鉴定和详细注释。分析发现,桔小实蝇蜕皮激素合成通路基因BdCyp302α1、BdCyp315α1和BdCyp314α1均属于"Halloween"基因家族,具有细胞色素氧化酶P450所特有签名序列：Helix-I、 Helix-K、底物识别性位点和血红蛋白结合位点等结构。桔小实蝇的BdCyp302α1和BdCyp314α1属于线粒体型,而BdCyp315α1属于微粒体细胞色素氧化酶。在蜕皮激素信号通路基因的研究中发现,桔小实蝇BdEcR-B1具有典型核受体保守结构域和微结构域,如DNA结合域和配体结构域等结构,双翅目昆虫EcR-B1亚型所特有的K/RRRW、S-rich、DL-rich、EESST/SEVV/TSS等特征基序。与BdEcR-B1相同,BdE75同属于核受体基因家族,具有其典型的配体结合域以及DNA结合域,其内含量2个C4锌指结构,在蜕皮激素信号通路上起初级响应元件的作用。BdL63序列N端具有cyclin-dependent kinase结构。BdTaiman属于p160核受体共激活子家族,具有basic helix-loop-helix (bHLH)结构域及乙酰化典型结构基序LxxLL。
2桔小实蝇蜕皮激素合成与信号通路基因的表达模式分析
2.1桔小实蝇蜕皮激素合成与信号通路基因在不同发育阶段的表达模式
在成功建立桔小实蝇蜕皮激素合成与信号通路基因的qPCR反应体系的基础上,以a-Tubulin为内参基因,分别对桔小实蝇蜕皮激素合成通路(BdNvd、 BdCyp306α1、BdCyp302α1、BdCyp315α1和BdCyp314α1)和信号通路(BdEcR-B1、 BdUSP、BdE75、BdL63、BdTaiman)基因在其不同发育阶段(幼虫、蛹和雌成虫),共25个时间点的表达情况进行了分析。
桔小实蝇蜕皮激素合成通路基因在不同日龄幼虫中的表达模式较为相似,BdNvd、BdCyp302α1、BdCyp314α1基因的最高表达量均出现在末龄幼虫阶段,与即将出现的化蛹行为有关。在蛹期,与BdNvd在蛹末期高表达不同,BdCyp306α1、 BdCyp302α1和BdCyp315α1基因相对最高表达量出现在化蛹初期,并随着蛹日龄的增加而显著地降低；BdCyp314α1则在蛹期呈现3个表达高峰,可能与20E介导的组织重建过程相关。在成虫期,除BdCyp314α1外,桔小实蝇蜕皮激素合成通路基因的最低表达量均出现在羽化初期,在第7日龄中出现显著性升高,并随即下降后再次上升,并在第19日龄成虫中达到相对表达量的最大值,表明蜕皮激素合成通路基因的表达与其性成熟和产卵行为有关。
就蜕皮激素信号通路基因而言,BdEcR-B1、BdUSP、BdL63和BdTaiman的表达量在末龄幼虫阶段均出现上升,暗示这些基因可能与蜕皮激素介导的变态反应有关。BdE75在蛹中期特异性高表达,可能在组织重建中具有重要作用。其余基因在蛹中后期表达模式比较相似,呈现逐渐下降的趋势,暗示由20E调控的组织重建过程也趋于完成。在成虫期,BdEcR-B1和BdE75基因的相对表达量随日龄的增加而出现上升；BdUSP和BdL63基因的相对表达量在前16日中保持相对稳定,在第19日龄出现明显的上升；而BdTaiman基因的相对最高表达量出现在雌虫羽化的第1天,并在随后的阶段保持相对稳定,推测上述基因在雌虫卵巢成熟过程中起不同作用。
2.2桔小实蝇蜕皮激素合成与信号通路基因在不同组织的表达模式
同样以a-Tubulin为内参基因,对上述基因在桔小实蝇5日龄幼虫前胸腺(复合体)、脂肪体、中肠、马氏管、表皮和气管的mRNA的表达水平进行了评价。结果发现,前胸腺仍为蜕皮激素早期合成的主要场所。BdNvd、BdCyp306α1和BdCyp315α1基因在前胸腺中特异性高表达,极显著高于脂肪体、中肠、马氏管、表皮和气管,而这些组织内的表达量无显著性差异。BdCyp302α1基因在脂肪体、前胸腺、马氏管／表皮、中肠／气管中的表达量逐步降低。而BdCyp314α1基因在中肠、马氏管、脂肪体中的相对表达量依次降低,但均显著性高于表皮、气管和前胸腺。说明桔小实蝇不同组织均参与蜕皮激素的合成,前胸腺、脂肪体和中肠是20E合成的主要场所：此外,马氏管除主要负责排泄外,也参与了蜕皮激素的合成。
在桔小实蝇蜕皮激素信号通路基因的研究中发现,BdEcR-B1和BdE75的表达模式较为相似,在脂肪体的表达量极显著高于其余5个组织,且在5个组织内无显著性差异；其中,BdE75在脂肪体内特异性高表达,其表达量是表皮的122倍。BdUSP在前胸腺表达量与脂肪体无显著性差异,但均极显著高于其他4个组织。而BdL63的表达量在脂肪体、前胸腺和马氏管中依次降低,而在马氏管、中肠和表皮中无显著性差异。BdTaiman基因在马氏管中的表达量显著性高于表皮,但与前胸腺、脂肪体和中肠差异不显著。综上,蜕皮激素信号通路基因主要集中在脂肪体内表达,由此推测,这可能与脂肪体营养储存和能量代谢功能相关。
3饥饿对桔小实蝇幼虫发育和蜕皮激素合成与信号通路基因的影响
3.1饥饿对桔小实蝇幼虫发育的影响
对桔小实蝇5日龄幼虫进行不同时长饥饿处理后发现,饥饿有效缩短幼虫历期,造成提前化蛹,且蛹体较小的现象,但并未引起幼虫存活率的降低,说明桔小实蝇在末龄幼虫的第1天,已经达到其化蛹所需最低体重阈值；在食物不足的情况下,通过提前变态成小个体蛹,来避免外界不利环境对自身的影响,维持种群延续。
3.2桔小实蝇蜕皮激素合成信号通路基因在饥饿胁迫下的表达模式
利用实时定量荧光PCR检测发现,其蜕皮激素合成通路基因表达水平在饥饿处理后出现明显的变化。BdNvd、BdCyp302α1和BdCyp314α1基因的相对表达量在饥饿胁迫6h后即出现显著性上调,其中BdCyp302α1和BdCyp314α1基因在24h达到极显著上调水平：而饥饿后48h后,BdNvd、BdCyp302α1和BdCyp314α1的表达水平出现显著性降低。对信号通路基因的研究发现,除BdL63外,其余各基因表达均出现显著性上调。由此推测,桔小实蝇幼虫在食物缺乏的状况下,可能通过提高蜕皮激素合成和信号通路基因的转录水平使体内蜕皮激素含量升高,信号传导加强,导致其幼虫提前化蛹,从而避免不良环境的胁迫。
4外源20E对桔小实蝇幼虫发育和蜕皮激素合成与信号通路基因的影响
4.1外源20E对桔小实蝇幼虫发育的影响
利用微量注射法,观察不同剂量20E处理对桔小实蝇幼虫发育的影响。结果发现,注射激素后,幼虫化蛹时间提前。同时,化蛹过程出现异常,并出现头部缢缩、黑化以及幼虫形态蛹的现象,且畸形的蛹宽和蛹长出现变化。畸形率和蛹死亡率显著上升。说明外源激素干扰了桔小实蝇幼虫正常生长发育和变态过程。
4.2外源20E对桔小实蝇蜕皮激素合成与信号通路基因的影响
经0.1μg/头剂量处理后,桔小实蝇蜕皮激素合成通路基因BdNvd、 BdCyp306α1、BdCyp302α1和BdCyp315α1均表现出相似的诱导反应,其相对表达量出现极显著上调,而BdCyp314α1的表达却被外源激素所抑制。桔小实蝇蜕皮激素信号通路基因的表达也表现出类似的诱导效应。除BdEcR-B1仅在处理1h后出现极显著的下调外,其它基因较对照而言,均出现极显著的上调。
经0.1和1.0μg/头外源20E处理后,BdNvd、BdCyp306α1和BdCyp315α1的表达量均极显著高于对照组；BdCyp302α1的表达量仅在0.1μg/头的剂量处理下极显著性高于对照组；而BdCyp314α1的表达量在0.5和1.0μg/头处理组的表达量均显著性低于对照。此外,BdEcR-B1和BdUSP均出现极显著上调。BdE75和BdL63对20E处理的反应较为一致,均在0.1μg和1.0μg/头处理后出现极显著性的上调。但0.5μg/头处理对蜕皮激素合成和信号通路基因的表达均表现出不同程度的抑制作用,该剂量可能是桔小实蝇体内某些生理反应的一个阈值,影响其生长发育等重要过程。
5桔小实蝇BdEcR-B1基因的功能研究
通过体外转录法获得高质量dsRNA,借助微量注射技术将外源dsRNA导入桔小实蝇5日龄幼虫体内。qPCR分析结果表明,BdEcR-B1基因表达量在注射72h后出现明显的下调,其表达量仅为对照组的53.4%。对桔小实蝇蜕皮激素信号通路基因的相对表达量进行分析发现,在靶基因沉默后,BdUSP和BdTaiman基因相对表达量出现了显著性下调,其中BdTaiman基因相对表达量在注射24-72h后出现极显著的下调,仅为对照组的9.7-12.5%。而BdE75基因和BdL63基因的转录并未受影响,出现2倍左右的上调。说明BdEcR-B1在蜕皮激素信号传导中直接调控其配体和共激活子的表达。
综上所述,本研究通过高通量测序、RT-PCR和RACE技术,分离克隆获得桔小实蝇蜕皮激素合成和信号通路基因cDNA全长序列,借助qPCR手段分析其在不同发育阶段、不同组织、饥饿胁迫以及激素处理条件下的表达模式,明确其调控机制和表达规律。同时,利用生测技术,研究饥饿和外源20E对桔小实蝇幼虫发育的影响。在此基础上,进一步利用RNAi技术,验证BdEcR-B1在蜕皮激素信号级联反应中的中心地位。研究结果有助于阐明蜕皮激素对昆虫发育和变态的调控机制,加深蜕皮激素合成与信号基因功能的认识,并为以上述通路基因为靶标的害虫防治方法提供理论依据和实践价值。
The oriental fruit fly, Bactrocera dorsalis (Hendel)(Diptera, Tephritidae) is a world-wide devastating agricultural pest. The adults lay eggs underneath the exocarp, and the larvae hatched and fed on the fruit, causing direct fruit damage and economic loss. Moreover, due to the wide host ranges, high adaptive capacity, fecundity and dispersal ability, B. dorsalis is documented on the quarantine target lists in many countries and treated as an export barrier in the international trade. Insects lack the de novo pathway of synthesizing20-hydroecdysone (20E), instead,20E is synthesized from dietary cholesterols or phytosterols. The mechanism involved in ecdysone biosynthesis is mediated by several P450enzymes, encoded by the Halloween gene family.20E, the most active form of ecdysteroid, acts as a critical hormone signal to coordinate insect embryonic development, larval growth and metamorphosis, pupal remodeling, courtship and reproduction precisely during the whole biological processes. The ecdysone receptor (EcR) could be induced by20E, when the expression level of EcR exceeds the threshold, EcR was assembled with ultraspiracle (USP) into a functional heterodimer receptor. After being assembled, the20E-EcR/USP complex sets off a series of early and late cascade responses, regulating cell proliferation, differentiation, or even cell programmed death. With RNAi technology's emerging and maturing, the gene functional study stepped into a new era. It is a very powerful tool in biological functional study, pharmacological screening, genetic therapy, pest management et al, which has greatly advanced the research of molecular biology and may have a prosperous future.
This dissertation focused on the ecdysone synthesis and signaling pathway of B. dorsalis, an economically important citrus pest. Firstly, the aforementioned genes involved in these two pathways were cloned, identified and detailed annotated using RT-PCR and RACE technologies as well as bioinformatics software. Secondly, the expression profiles of these genes were analyzed in different developmental stages and tissues. Bioassay and qPCR were further applied to evalute the effect of starvation in biological and molecular responses under nutrient stress. Insect growth regulators were often used in pest control, in the purpose of unbalancing the endocrinal signal of insects, causing its death. The impacts on the growth, metamorphosis of the larvae and expression profiles of aforementioned genes were tested after injected with exogenous20E. Finally, the function of BdEcR-Bl in ecdysone signaling cascade was interpreted by RNAi. The study on molecular regulation mechnism of metamorphsis and development, especially the ecdysone synthesis and signaling pathways, could deepen the understanding of function and regulation mechanism of the genes in the fields, providing potentional tagrets and theoretical basis in transgenic food applying and new insectcides screening in pest control. The main results are as follows:
1Molecular cloning and characterization of ecdysone synthesis and signaling pathway genes of B. dorsalis
Based on the high-throughput transctiptome sequencing of B. dorsalis,7novel genes were isolated with RT-PCR and RACE technologies, and deposited in GenBank with the following names and accession numbers:BdCyp302α1(JQ027284), BdCyp315α1(KC515377), BdCyp314α1(JQ229645), BdEcR-B1(JQ034623), BdE75(KC515378), BdL63(KC515379) and BdTaiman (JQ266268). The open reading frames (ORF), pupative amino acids sequences, physico-chemical properties, transmembrane regions and conserved domains were predicted and annotated in detail. The results indicated that BdCyp302α1, BdCyp315α1and BdCyp314α1in ecdysone synthesis pathway, were the members of Halloween gene family, and harbored the typical signature motifs of P450enzymes, such as Helix-I, Helix-K, substrate recognition site (SRS) and heme-binding domains. BdCyp302α1and BdCyp314α1were classified into mitochondrial, while BdCyp315α1was classified into microsomal type of P450gene. BdEcR-B1contained several typical nuclear receptor structures, such as DNA-binding domain (DBD), ligand-binding domain (LBD), and EcR-B1isoform-specific motifs like K/RRRW, S-rich, DL-rich, EESST/SEVV/TSS, which especially contained in Diptera. BdE75also belonged to nuclear receptor family with DBD, LBD and two zinc finger motifs, acting as an early response element in ecdysone signaling cascade. At the N-terminus, BdL63possessed a cyclin-dependent kinase domain. Besides, BdTaiman was identified to be a member of p160family of nuclear receptor coactivator, harboring basic helix-loop-helix (bHLH) and classical acetylation motif LxxLL.
2Expression patterns of ecdysone synthesis and signaling pathway genes of B. dorsalis
2.1Expression patterns of ecdysone synthesis and signaling pathway genes in different stages
Total RNA of different developmental stages was extracted from the larvae (1-day-old to8-day-old with one-day interval), pupae (1day-old to10day-old with one-day interval) and female adults (1day-old to19day-old with three-day interval) of B. dorsalis. On the one hand, the expression profiles of5genes functioning in ecdysone synthesis pathway, BdNvd, BdCyp306α1, BdCyp302al, BdCyp315al, BdCyp314α1and5signaling pathway genes, BdEcR-B1, BdUSP, BdE75, BdL63and BdTaiman were estimated in those samples by qPCR, using a-Tubulin as an internal control. The results showed that, during the larval stages, BdNvd, BdCyp302al and BdCyp314al shared the same profile patterns, with the highest expression levels occurred in the mature larvae, suggesting they might participate in the following pupation course. During the pupal stages, BdCyp306al, BdCyp302α1and BdCyp315al were highly expressed at the very beginning of this stage, and declined significantly with the increase of time, different from the expression pattern of BdNvd. The relative expression of BdCyp314α1was demonstrated with3peaks during the pupal stage, implying it might play an important role in tissue remodeling. In newly emerged adults, the relative expressions levels of BdNvd, BdCyp306α1, BdCyp302α1and BdCyp315al were the lowest, and significantly rised at the seventh day, then decreased and rised gradually, reached the maxium level at the19-day-old female, indicating they could involve in sex maturation and oviposition.
On the other hand, ecdysone signaling pathway genes BdEcR-B1, BdUSP, BdL63and BdTaiman were highly transcribed during the late larval stage, involving in coordinating the ecdysone signal with metamorphosis at this stage. BdE75was highly expressed in the middle pupal stage, implying its key role in tissue remodeling. The other signaling pathway genes shared the similar expression patterns during pupal stage, which decreased gradually during the middle-late couse, suggesting the procedure of adult morphogenesis tend to be complete under the regulation of20E. After eclosion, the expression levels of BdEcR-B1and BdE75gradually rised; BdUSP and BdL63significantly rised at the19th day. Besides, BdTaiman highly expressed in the1-day-old females, and remained stable in the following fifteen days, implying their different roles in ovary maturation.
2.2Expression patterns of ecdysone synthesis and signaling pathway genes in different tissues
The relative expression levels of ecdysone synthesis and signaling pathway genes were evaluated in six tissues including prothoracic glands (mixture), fatbody, midgut, Malpighian tubules, integument and trachea, which were dissected from the5-day-old larvae of B. dorsalis, with a-Tubulin as a reference gene. The results revealed that the prothoracic glands remain to be the main site which involved in early step of20E synthesis. BdNvd, BdCyp306α1and BdCyp315α1very highly expressed in prothoracic glands, and their expression levels showed no significant difference in fatbody, midgut, Malpighian tubules, integument and trachea. The expression profiles of BdCyp302α1were successively decrease in fatbody, prothoracic glands, Malpighian tubules/integument and midgut/integument. Furthermore, the expression levels of BdCyp314α1were highly expressed in midgut, Malpighian tubules and fatbody. The results indicated that, although many tissues participated in ecdysone synthesis, prothoracic glands, fatbody and midgut remained to be the main sites for the process. In addition to excretion, Malpighian tubules were also in charge of ecdysone biosynthesis.
The expression levels of signaling pathway genes BdEcR-B1and BdE75shared the same tissue distribution pattern, whose expression levels in the fatbody were relatively higher than those in the other five tissues. In additin, BdE75was exclusively highly expression in fatbody. The mRNA expression levels of BdUSP showed no significant differences in prothoracic glands and midgut, but sigfinficantly higher than those in midgut, Malpighian tubules, integument and trachea. The expression profiles of BdL63were successively decrease in fatbody, prothoracic glands and Malpighian tubules, however, there were no difference among Malpighian tubules, midgut and integument. BdTaiman showed a higher expression level in the Malpighian tubules than in integument, but no significant difference between Malpighian tubules, fatbody and midgut. The results showed that the20E signaling pathway genes predominately expressed in the fatbody, indicating those genes might be involved in nutrients storage and energy utilization of this certain tissue.
3The effects of starvation on larvae development and expression profiles of ecdysone synthesis and signaling pathway genes of B. dorsalis
3.1The effects of starvation on the larvae development of B. dorsalis
Different food deprivation time were applied to the5-day-old larvae to illustrate the stress response of B. dorsalis. The data showed that starvation could shorten the larval duration, causing prepupation, but no side effect on survival rate, indicated the body weight of the5-day-old larvae, the first day of the third instar, exceed the thredhold value of pupation. Moreover, when food was insufficient, the larvae metamorphosis precociously, avoiding harmful environmental stress.
3.2Expression patterns of ecdysone synthesis and signaling pathway genes under starvation stress
In addition to bioassay, qPCR was applied to illustrate the stress response of ecdysone synthesis and signaling pathway genes under starvation. The expressions levels of BdNvd, BdCyp302al and BdCyp314al were signicantly elevated at6h post food deprivation, and BdCyp302al and BdCyp314al were very highly elevated at24h. At48h, the expressions of BdCyp302α1and BdCyp314α1were significantly lower than that of the control. Except for BdL63, ecdysone signaling pathway genes were all significantly up-regulated. These results suggested that nutritional deprivation could cause a rapid rise of20E titer and strengthened signals by up-regulating the expression levels of ecdysone synthesis and signaling pathway genes, which would ultimately lead to pupate precociously, avoiding the detrimental effects of the adverse environmental stress.
4The effects of exogenous20E on larvae development and expression profiles of ecdysone synthesis and signaling pathway genes of B. dorsalis
4.1The effects of exogenous20E on the larvae development of B. dorsalis
This study has been carried out to investigate the effects of exogenous20E on the5-day-old larvae of B. dorsalis by injecting different doses of20E. The results showed that20E caused precocious pupation, extraordinary phenomena, including abnormal pupation in artificial diet, shrunken, melanized and larval-formed pupae. The length, width and malformation rate were significantly different between the control and treatment. The results also showed that there may be a positive correlation between mortality and the dose of20E, which indicating that the exogenous20E had a remarkable negative effect on the growth and metamorphosis of B. dorsalis.
4.2The effects of exogenous20E on expression profiles of ecdysone synthesis and signaling pathway genes of B. dorsalis
The expressions of ecdysone synthesis pathway genes BdNvd,BdCyp306α1, BdCyp302al and BdCyp315α1showed a similar induced effect after injection with0.1μg/insect of exogenous20E, which were increased significantly. However, the expression of BdCyp314α1was depressed by exogenous20E. The expression profiles of ecdysone signaling pathway genes showed the same induced effects, which were highly induced, except for BdEcR-B1at1h after injection.
After injection with0.1and1.0μg/insect of exogenous20E, the expression levels of BdNvd, BdCyp306α1and BdCyp315α1were significantly elevated compared to the control, but BdCyp302α1was highly upregulated by0.1Lg20E. However, the expression of BdCyp314α1was significantly down-regulated after injection with0.5and1.0μg/insect of20E. In addition, BdEcR-B1and BdUSP were up-regulated. The expressions of BdE75and BdL63were increased by injection with0.1and1.0μg/insect20E. However, the0.5μg/insect of20E suppressed the expressions of both ecdysone synthesis and signaling pathway genes in varying dgree, indicating this could be a threshold dose in some important physiological reaction, intefering the development of B. dorsalis.
5Functional study of BdEcR-B1of B. dorsalis
High quality of dsRNA was synthesized by in vitro transcription, and delivered into B. dorsalis by microinjection. Quantitative PCR showed that, the expression level of BdEcR-B1was reduced significantly compared to dsGFP treatement after injected for72h, and so did in BdUSP and BdTaiman. Especially, the expression of BdTaiman was depressed to a very low level, down-regulated87.5-90.3%compared to the control. However, BdE75and BdL63showed2-fold up-regulated after injection. The results indicated that BdEcR-Bl play an important role in controlling the expression levels of BdUSP and BdTaiman, which worked as its heterodimer partner and coactivator in ecdysone signaling pathway.
Taken together, the full-length of ecdysone synthesis and signaling pathway genes were obtained by high-throughput sequencing, RT-PCR and RACE methods. Quantitative PCR was applied to illustrate the expression patterns of these genes in different stages and tissues, and under starvation stress and hormone interference conditions. Meanwhile, bioassay strategy was utilized to illuminate the response to starvation and exogenous20E on larvae development. Further, based on RNAi, the central role of BdEcR-B1in ecdysone signal transduction was confirmed in B. dorsalis. All these results will not only elucidate the molecular mechanism of ecdysone regulation on growth and metamorphosis, deepen our understanding the functions of the genes in ecdysone synthesis and signaling pathway, but also provide theoretical basis for EcR-targeting pest control.
引文
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