Molecular cloning of ecdysone 20-hydroxylase and expression pattern of the enzyme during embryonic development of silkworm Bombyx mori.
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.
Maeda
Sayaka
S
Department of Biology, Konan University, Kobe 658-8501, Japan.
Nakashima
Asuka
A
Yamada
Ryouichi
R
Hara
Noriyuki
N
Fujimoto
Yoshinori
Y
Ito
Yoichi
Y
Sonobe
Haruyuki
H
D000595:Amino Acid Sequence; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry / Q000235:genetics*; D016367:Baculoviridae; D001483:Base Sequence; D012831:Bombyx / Q000196:embryology* / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D047108:Embryonic Development; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010802:Phylogeny; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013250:Steroid Hydroxylases / Q000737:chemistry / Q000235:genetics*
D018076:DNA, Complementary; D012333:RNA, Messenger; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C020095:ecdysone 20-hydroxylase