Molecular cloning and characterization of Ecdysone oxidase and 3-dehydroecdysone-3α-reductase involved in the ecdysone inactivation pathway of silkworm, Bombyx mori.
Molting hormone (ecdysteroid) is one of the most important hormones in insects. The synthesis and inactivation of the ecdysteroid regulate the developmental process of insects. A major pathway of ecdysone inactivation is that ecdysone is converted to 3-dehydroecdysone, and then further to 3-epiecdysone in insects. Two enzymes (ecdysone oxidase: EO and 3DE-3α-reductase) participate in this pathway. In this study, based on the previously characterized cDNAs in Spodoptera littoralis, we cloned and characterized EO and 3DE-3α-reductase genes in the silkworm, Bombyx mori. The heterologously expressed proteins of the two genes in yeast showed the ecdysone oxidase and 3DE-3α-reductase activities, respectively. Expression of BmEO was only detected in the midgut at transcriptional and translational levels. We also localized EO within the midgut goblet cell cavities. For Bm3DE-3α-reductase gene, RT-PCR and western blot showed that it was expressed in the midgut and the Malpighian tubules. Moreover, we localized 3DE-3α-reductase within the midgut goblet cell cavities and the cytosol of principal cells of the Malpighian tubules. These two genes have similar expression profiles during different developmental stages. Both genes were highly expressed at the beginning of the 5th instar, and remained a relative low level during the feeding stage, and then were highly expressed at the wandering stage. All these results showed that the profiles of the two genes were well correlated with the ecdysteroid titer. The functional characterization of the enzymes participating in ecdysone inactivation in the silkworm provides hints for the artificial regulation of the silkworm development and biological control of pests.
The Key Sericultural Laboratory of Agricultural Ministry, Southwest University, Chongqing, 400715, China.
Wei
W
Sun
Yi-Hong
YH
Shen
Deng-Wei
DW
Qi
Zhong-Huai
ZH
Xiang
Ze
Z
Zhang
D015096:3-Hydroxysteroid Dehydrogenases / Q000032:analysis / Q000737:chemistry / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D012831:Bombyx / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D003001:Cloning, Molecular; D008969:Molecular Sequence Data; D010802:Phylogeny; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein
D015096:3-Hydroxysteroid Dehydrogenases; C104053:3-dehydroecdysone 3alpha-reductase; C020096:ecdysone oxidase