cDNA cloning and characterization of Bombyx mori juvenile hormone esterase: an inducible gene by the imidazole insect growth regulator KK-42.
The insect growth regulator (IGR) imidazole KK-42 induces hemolymph juvenile hormone esterase activity and precocious metamorphosis in Bombyx mori. As an initial step to understand the molecular action of KK-42, we isolated a full-length of juvenile hormone esterase cDNA from B. mori (BmJHE). The deduced amino acid sequence of BmJHE showed high identity to JHEs of Heliothis virescens (54%) and Choristoneura fumiferana (52%). Recombinant BmJHE protein expressed in the baculovirus expression system hydrolyzed 3H-JH III and JH analog, HEPTAT, indicating that BmJHE cDNA encodes functional JH esterase. Northern blot analysis showed that the BmJHE transcript was present predominantly in the fat body at the beginning of the last larval instar. During this instar, BmJHE transcript increased gradually until day 7, then decreased, and increased again on day 10 in the fat body. This temporary expression pattern was similar to that of JHE enzyme activity in hemolymph. In contrast, in the 4th instar, the BmJHE transcript was present in the fat body even though hemolymph JHE activity was very low. Western blot analysis using anti-BmJHE antiserum showed BmJHE protein was present in hemolymph during the 5th instar but not during the 4th instar. These results indicate that BmJHE protein is secreted into hemolymph at the metamorphic stage. Hemolymph JHE activity was high in precociously metamorphosed 4th instar larvae (treated KK-42) but low in normal 4th and extra-molted 6th instar larvae (fed 20E). KK-42-treated larvae showed high expression level of BmJHE transcript in the fat body, suggesting that KK-42 enhances BmJHE gene expression in the fat body.
Hirai
M
M
National Institute of Agrobiological Science, 1-2 Owashi, Tsukuba, Ibaraki 305-8634, Japan.
Kamimura
M
M
Kikuchi
K
K
Yasukochi
Y
Y
Kiuchi
M
M
Shinoda
T
T
Shiotsuki
T
T
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae; D001483:Base Sequence; D012831:Bombyx / Q000201:enzymology* / Q000235:genetics; D002265:Carboxylic Ester Hydrolases / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004441:Ecdysterone / Q000494:pharmacology; D015870:Gene Expression; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D006458:Hemolymph / Q000201:enzymology; D007093:Imidazoles / Q000494:pharmacology*; D007303:Insect Vectors; D007605:Juvenile Hormones / Q000494:pharmacology*; D008969:Molecular Sequence Data; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution; D015533:Transcriptional Activation
D018076:DNA, Complementary; D007093:Imidazoles; D007605:Juvenile Hormones; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D004441:Ecdysterone; C063652:1-benzyl-5-(2,6-dimethyl-1,5-heptadienyl)imidazole; D002265:Carboxylic Ester Hydrolases; C038003:juvenile hormone esterase