Molecular cloning and characterization of hemolymph 3-dehydroecdysone 3 beta-reductase from the cotton leafworm, Spodoptera littoralis - A new memberof the third superfamily of oxidoreductases

The primary product of the prothoracic glands of last instar larvae of Spod optera littoralis is 3-dehydroecdysone (3DE). After secretion, 3DE is reduc ed to ecdysone by 3DE 3 beta-reductase in the hemolymph, We have previously purified and characterized 3DE 3 beta-reductase from the hemolymph of S, l ittoralis, In this study, cDNA clones encoding the enzyme were obtained by reverse transcription-polymerase chain reaction, employing primers based on the amino acid sequences, in conjunction with 5'- and 3'-rapid amplificati on of cDNA ends. Multiple polyadenylation signals and AT-rich elements were found in the 3'-untranslated region, suggesting that this region may have a role in regulation of expression of the gene. Conceptual translation and amino acid sequence analysis suggest that 3DE 3 beta-reductase from S, litt oralis is a new member of the third superfamily of oxidoreductases, Norther n analysis shows that 3DE 3 beta-reductase mRNA transcripts are widely dist ributed, but are differentially expressed, in some tissues. The development al profile of the mRNA revealed that the gene encoding 3DE 3 beta-reductase is only transcribed in the second half of the last larval instar and that this fluctuation in expression accounts for the change in the enzyme activi ty during the instar, Southern analysis indicates that the 3DE 3 beta-reduc tase is encoded by a single gene, which probably contains at least one intr on.