Molecular cloning of ecdysone 20-hydroxylase and expression pattern of the enzyme during embryonic development of silkworm Bombyx mori. Comp Biochem Physiol B Biochem Mol Biol
In various insects, 20-hydroxyecdysone (20E) is indispensable for embryonic development. In eggs of the silkworm Bombyx mori, 20E has been demonstrated to be produced by two metabolic pathways: de novo synthesis from cholesterol and dephosphorylation of ovary-derived physiologically inactive ecdysteroid phosphates. In the former, ecdysone 20-hydroxylase (E20OHase) has been suggested to be a key enzyme. In the latter, it has been demonstrated that the dephosphorylation of ecdysteroid phosphates is catalyzed by a specific enzyme, ecdysteroid-phosphate phosphatase (EPPase). In this study, a cDNA encoding E20OHase was cloned from 3-day-old nondiapause eggs of B. mori and sequenced using PCR techniques. The protein exhibited the signature sequences characteristic of P450 enzymes, and mediated the conversion of ecdysone to 20E using the baculovirus expression system. Semi-quantitative analysis revealed that the E20OHase mRNA is expressed predominantly during gastrulation and organogenesis in nondiapause eggs, but is scarcely detected in diapause eggs whose development is arrested at the late gastrula stage. The developmental changes in the expression patterns of E20OHase and EPPase suggest that both enzyme activities are regulated at the transcription level, and both enzymes contribute cooperatively to 20E formation during embryonic development.
Available from: Heleen Verlinden
- "CYP307A1 and CYP307A2 probably act in the above described 'Black Box' but their exact biochemical function remains unknown (Ono et al., 2006). Since their first functional identification in D. melanogaster, a similar function for these Halloween genes has also been described in two lepidopteran species: the silkworm Bombyx mori (Maeda et al., 2008; Niwa et al., 2004, 2005; Warren et al., 2004) and the tobacco hornworm, Manduca sexta (Rewitz et al., 2006a,b). Halloween genes have also been described/predicted in several other Arthropod species: in another lepidopteran, the cotton leafworm, Spodoptera littoralis (Iga and Smagghe, 2010), but also in the dipteran Aedes aegypti (Sieglaff et al., 2005), the hemipteran pea aphid Acyrthosiphon pisum (Christiaens et al., 2010) and the crustacean Daphnia pulex (Rewitz and Gilbert, 2008). "
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ABSTRACT: The functional characterization of the Halloween genes represented a major breakthrough in the elucidation of the ecdysteroid biosynthetic pathway. These genes encode cytochrome P450 enzymes catalyzing the final steps of ecdysteroid biosynthesis in the dipteran Drosophila melanogaster and the Lepidoptera Manduca sexta and Bombyx mori. This is the first report on the identification of two Halloween genes, spook (spo) and phantom (phm), from a hemimetabolous orthopteran insect, the desert locust Schistocerca gregaria. Using q-RT-PCR, their spatial and temporal transcript profiles were analyzed in both final larval stage and adult locusts. The circulating ecdysteroid titers in the hemolymph were measured and found to correlate well with changes in the temporal transcript profiles of spo and phm. Moreover, an RNA interference (RNAi)-based approach was employed to study knockdown effects upon silencing of both transcripts in the fifth larval stage. Circulating ecdysteroid levels were found to be significantly reduced upon dsRNA treatment.
Available from: Ling Gan
- "Antennal binding protein and chemosensory protein are responsible for sensory signaling and might be required for the correct recognition of some odors [24-26]. Our analysis detected the expression of five cytochrome P450 genes in cluster I, none of which are silkworm halloween genes, which are involved in biosynthesis of 20-hydroxyecdysone (20E) from cholesterol [27-32]. Since the cytochrome P450 gene superfamily consists of 86 putative members , the expression of these five cytochrome P450 genes in silkworm brain might provide clues for their functional study. "
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ABSTRACT: Molecular genetic studies of Bombyx mori have led to profound advances in our understanding of the regulation of development. Bombyx mori brain, as a main endocrine organ, plays important regulatory roles in various biological processes. Microarray technology will allow the genome-wide analysis of gene expression patterns in silkworm brains.
We reported microarray-based gene expression profiles in silkworm brains at four stages including V7, P1, P3 and P5. A total of 4,550 genes were transcribed in at least one selected stage. Of these, clustering algorithms separated the expressed genes into stably expressed genes and variably expressed genes. The results of the gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analysis of stably expressed genes showed that the ribosomal and oxidative phosphorylation pathways were principal pathways. Secondly, four clusters of genes with significantly different expression patterns were observed in the 1,175 variably expressed genes. Thirdly, thirty-two neuropeptide genes, six neuropeptide-like precursor genes, and 117 cuticular protein genes were expressed in selected developmental stages.
Major characteristics of the transcriptional profiles in the brains of Bombyx mori at specific development stages were present in this study. Our data provided useful information for future research.
Available from: Guy Smagghe
- "The products of phm, dib and sad sequentially convert the precursor of ecdysone, 2,22,25-trideoxyecdysone (ketodiol), to 22,2-dideoxyecdysone (ketotriol), 2-deoxyecdysone and ecdysone      . Further on, the product of shd converts ecdysone to 20E   . The initial conversion process from 7-dehydrocholesterol to ketodiol has not been characterized and remains as the 'Black Box'. "
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ABSTRACT: 20-Hydroxyecdyone (20E), an active form of ecdysteroid, is the key hormone in insect growth and development. The biosynthesis of ecdysteroid is triggered and under the control of the neuropeptide, prothoracicotropic hormone (PTTH). To date, five cytochrome P450 enzymes, namely Spook (Spo), Phantom (Phm), Disembodied (Dib), Shadow (Sad) and Shade (Shd) related to ecdysteroid biosynthesis, are identified and the character of last four enzymes is well studied in Drosophila melanogaster, Bombyx mori and Manduca sexta. These genes are called Halloween genes and mediate the biosynthesis of 20E from cholesterol. In this study, we extended these works to a major pest insect in agriculture, the cotton leafworm Spodoptera littoralis (Lepidoptera: Noctuidae). We identified the sequence of five Halloween genes, and the converted amino acid sequences were compared with those of other insects. The phylogenetic analysis clearly showed separated clusters of each gene and the evolutional conservation in insects with a high similarity in Lepidoptera. Spo, phm, dib and sad were predominantly expressed in prothoracic glands, and shd was expressed in fat body and Malpighian tubules at the last instar larvae. Spo expression was kept high level between day 2 and day 4 after ecdysis. The expression of phm and dib peaked at day 2, and sad and shd expressions peaked at day 2 and day 4 after ecdysis. In addition, the hemolymph ecdysteroid titer showed a small peak at day 2 and a large peak at day 4 after ecdysis. These results suggest the importance of Halloween genes in ecdysone biosynthesis by prothoracic glands and conversion of ecdysone into 20E by fat body in larval-pupal metamorphosis.
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