Cloning and expression pattern of 3-dehydroecdysone 3β-reductase (3DE 3β-reductase) from the silkworm, Bombyx mori L.
Molting in insects is regulated by molting hormones (ecdysteroids), which are also crucial to insect growth, development, reproduction, etc. Ecdysone was inactivated to 3-dehydroecdysone (3DE) under ecdysone oxidase (EO), and followed by NAD(P)H-dependent irreversible reduction to 3-epiecdysteroid under 3DE 3a-reductase. On the other hand, 3-dehydroecdysone undergoes reversible reduction to ecdysone by 3DE 3β-reductase in the hemolymph. In this article, we cloned and characterized 3-dehydroecdysone 3β-reductase (3DE 3β-reductase) in the different tissues and the developing stage from the silkworm, Bombyx mori L. The B. mori 3DE 3β-reductase cDNA contains an ORF 972 bp and the deduced protein sequence containing 323 amino acid residues. Analysis showed that the deduced 3DE 3β-reductase belongs to the aldo-keto reductase (AKR) superfamily, which has the NAD(P)-binding domain, indicating that the function of 3DE 3β-reductase depends on the existence of NAD(P)H. Using Escherichia coli, a high level expression of a fusion polypeptide band of approx. 40 kDa was observed. The high transcription of 3DE 3β-reductase was mainly observed in the genitalia and fatty bodies in the third day of the fifth-instar larvae, followed next in the head, epidermis, and hemocytes. The expression of 3DE 3β-reductase in the early of every instar was lower than that in the late of instar. When the titer of 3DE is low, higher expression of 3DE 3β-reductase is necessary to maintain the ecdysone titer in body through converting 3DE to ecdysone, while the 3DE titer is high, the expression of 3DE 3β-reductase showed feedback inhibition.
Key Laboratory of Animal Epidemic Etiology and Immunological Prevention of Ministry of Agriculture, Zhejiang University, Hangzhou, People's Republic of China.
Hua-jun
HJ
Yang
Xin-ju
XJ
Liu
Fang
F
Zhou
Jia-biao
JB
Hu
Roy
R
Bhaskar
Xing-hua
XH
Li
Chun-guang
CG
Sun
Firdose Ahmad
FA
Malik
Yan-shan
YS
Niu
Yun-gen
YG
Miao
D015096:3-Hydroxysteroid Dehydrogenases / Q000378:metabolism*; D000429:Alcohol Oxidoreductases / Q000378:metabolism; D000449:Aldehyde Reductase; D000074408:Aldo-Keto Reductases; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D012831:Bombyx / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D004440:Ecdysone / Q000031:analogs & derivatives* / Q000378:metabolism; D025461:Feedback, Physiological; D018507:Gene Expression Regulation, Developmental; D019476:Insect Proteins / Q000235:genetics / Q000378:metabolism; D007814:Larva / Q000378:metabolism; D008969:Molecular Sequence Data
D018076:DNA, Complementary; D019476:Insect Proteins; D004440:Ecdysone; C047104:3-dehydroecdysone; D015096:3-Hydroxysteroid Dehydrogenases; D000429:Alcohol Oxidoreductases; D000074408:Aldo-Keto Reductases; D000449:Aldehyde Reductase; C103187:3-dehydroecdysone 3beta-reductase