| 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. dorsalisBased 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. dorsalis2.1Expression patterns of ecdysone synthesis and signaling pathway genes in different stagesTotal 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. dorsalis3.1The effects of starvation on the larvae development of B. dorsalisDifferent 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 stressIn 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. dorsalis4.1The effects of exogenous20E on the larvae development of B. dorsalisThis 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. dorsalisThe 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. dorsalisHigh 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. |
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