Article

Molecular cloning of ecdysone 20-hydroxylase and expression pattern of the enzyme during embryonic development of silkworm Bombyx mori.

Department of Biology, Konan University, Kobe 658-8501, Japan.
Comparative Biochemistry and Physiology Part B Biochemistry and Molecular Biology (Impact Factor: 1.9). 04/2008; 149(3):507-16. DOI: 10.1016/j.cbpb.2007.11.015
Source: PubMed

ABSTRACT 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.

0 Bookmarks
 · 
81 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: It was predicted that the genome of silkworm, Bombyx mori, has at least 79 P450 genes; however, P450 genes that are related to the catabolism of exogenous compounds were not reported. In this study we cloned two CYP4 (named CPY4M5 and CYP4M9) and four CYP6 (named CYP6AB5, CYP6AE9, CYP6AE22 and CYP6AU1) genes by using both bioinformatics and RT-PCR approaches. Sequence analysis showed that these genes contained conserved P450 gene sequence regions and one conserved intron. CYP4M5 and CYP4M9 genes were clustered together in a mode of "head-to-tail" possibly due to gene duplication. Blast analysis showed that these P450 genes shared significant similarity with CYP4 and CYP6 genes that are involved in the catabolism and detoxification of exogenous compounds in other insect species. RT-PCR results showed that these P450 genes were highly expressed in the midgut and fat body of B. mori. As the instar age increased, these P450 genes exhibit different expression patterns. When B. mori was exposed to 1.75 × 10(-5 )% of cypermethrin, 3.5 × 10(-6 )% of cypermethrin and 0.1 % of rutin, expression of CYP6AB5 was increased by 2.3-fold, 2.2-fold and 1.9-fold, respectively. Exposure of B. mori to 0.1 % quercetin does not change the expression of CYP6AB5. In contrast, expression of the other five P450 genes was inhibited after exposed to these compounds.
    Molecular Biology Reports 05/2014; · 1.96 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: We investigated here the ecdysteroid titers and the expression of six genes coding for known enzymes of the ecdysteroid biosynthesis in the testes of last instar larvae of the pest cotton leafworm, Spodoptera littoralis. We showed that the timing of the ecdysteroid profile was the same in testes and in hemolymph, with a small peak at day 2 and a large one at day 4 after ecdysis. Ecdysone and 20-hydroxyecdysone (20E) were detected in both tissues. 20E was the major ecdysteroid in testes and in hemolymph from day 4. Interestingly, the gene expression of the steroidogenetic enzymes, Neverland, and the five cytochrome P450 enzymes encoded by the Halloween genes was confirmed in the testes, and varied during the instar. However, from the data obtained so far, we cannot conclude that the measured ecdysteroids in the testes result from the activity of the genes under study. Indeed, it is suggested that the ecdysone produced centrally in the prothoracic glands, could have been transformed into 20E in the testes, where Sl-shade is well expressed.
    Archives of Insect Biochemistry and Physiology 09/2012; · 1.52 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Steroid hormones ecdysteroids regulate varieties of developmental processes in insects. Although the ecdysteroid titer can be increased experimentally with ease, its artificial reduction, although desirable, is very difficult to achieve. Here we characterized the ecdysteroid-inactivating enzyme ecdysteroid-22-oxidase (E22O) from the entomopathogenic fungus Nomuraea rileyi and used it to develop methods for reducing ecdysteroid titer and thereby controlling insect development. K(m) and K(cat) values of the purified E22O for oxidizing ecdysone were 4.4 μM and 8.4/s, respectively, indicating that E22O can inactivate ecdysone more efficiently than other ecdysteroid inactivating enzymes characterized so far. The cloned E22O cDNA encoded a FAD-dependent oxidoreductase. Injection of recombinant E22O into the silkworm Bombyx mori interfered with larval molting and metamorphosis. In the hemolymph of E22O-injected pupae, the titer of hormonally active 20-hydroxyecdysone decreased and concomitantly large amounts of inactive 22-dehydroecdysteroids accumulated. E22O injection also prevented molting of various other insects. In the larvae of the crambid moth Haritalodes basipunctalis, E22O injection induced a diapause-like developmental arrest, which, as in normal diapause, was broken by chilling. Transient expression of the E22O gene by in vivo lipofection effectively decreased the 20-hydroxyecdysone titer and blocked molting in B. mori. Transgenic expression of E22O in Drosophila melanogaster caused embryonic morphological defects, phenotypes of which were very similar to those of the ecdysteroid synthesis deficient mutants. Thus, as the first available simple but versatile tool for reducing the internal ecdysteroid titer, E22O could find use in controlling a broad range of ecdysteroid-associated developmental and physiological phenomena.
    Journal of Biological Chemistry 03/2012; 287(20):16488-98. · 4.60 Impact Factor