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: 1.92). 04/2008; 149(3):507-16. DOI:10.1016/j.cbpb.2007.11.015 pp.507-16
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.

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Keywords

20-hydroxyecdysone
 
3-day-old nondiapause eggs
 
B. mori
 
baculovirus expression system
 
cDNA encoding E20OHase
 
de novo synthesis
 
developmental changes
 
E20OHase mRNA
 
ecdysone 20-hydroxylase
 
ecdysteroid-phosphate phosphatase
 
embryonic development
 
enzyme activities
 
key enzyme
 
nondiapause eggs
 
ovary-derived physiologically inactive ecdysteroid phosphates
 
PCR techniques
 
signature sequences characteristic
 
silkworm Bombyx mori
 
specific enzyme
 
transcription level
 

Sayaka Maeda