CLONING AND EXPRESSION PATTERN OF 3-DEHYDROECDYSONE 3 beta-REDUCTASE (3DE 3 beta-REDUCTASE) FROM THE SILKWORM, Bombyx mori L.

摘要

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 3breductase in the hemolymph. In this article, we cloned and characterized 3-dehydroecdysone 3beta-reductase (3DE 3 beta-reductase) in the different tissues and the developing stage from the silkworm, Bombyx mori L. The B. mori 3DE 3 beta-reductase cDNA contains an ORF 972 bp and the deduced protein sequence containing 323 amino acid residues. Analysis showed that the deduced 3DE 3b-reductase belongs to the aldo-keto reductase (AKR) superfamily, which has the NAD(P)-binding domain, indicating that the function of 3DE 3 beta-reductase depends on the existence of NAD(P)H. Using Escherichia coli, ahigh level expression of a fusion polypeptide band of approx. 40 kDa was observed. The high transcription of 3DE 3 beta-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 3 beta-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 3 beta-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 3 beta-reductase showed feedback inhibition.