Cloning of putative odorant-degrading enzyme and integumental esterase cDNAs from the wild silkmoth, Antheraea polyphemus.
Odorant-degrading enzymes have been postulated to participate in the fast deactivation of insect pheromones. These proteins are expressed specifically in the sensillar lymph of insect antennae in such low amounts that, hitherto, isolation and protein-based cDNA cloning has not been possible. Using degenerate primers based on conserved amino acid sequences of insect carboxylesterases and juvenile hormone esterases, we were able to amplify partial cDNA fragments, which were then used for the design of gene-specific primers for RACE. This bioinformatics approach led us to the cloning of cDNAs, encoding a putative odorant-degrading enzyme (Apol-ODE) and a putative integumental esterase (Apol-IE) from the wild silkmoth, Antheraea polyphemus. Apol-ODE had a predicted molecular mass of 59,994 Da, pI of 6.63, three potential N-glycosylation sites, and a putative catalytic site Ser characterized by the sequence Gly(195)-Glu-Ser-Ala-Gly-Ala. Apol-IE gave calculated molecular mass of 61,694 Da, pI of 7.49, two potential N-glycosylation sites, and a putative active site with the sequence Gly(214)-Tyr-Ser-Ala-Gly. The transcript of Apol-ODE was detected by RT-PCR in male antennae and branches (sensillar tissues), but not in female antennae and other control tissues. Apol-IE was detected in male and female antennae as well as legs.
Ishida
Yuko
Y
Honorary Maeda-Duffey Lab, Department of Entomology, University of California Davis, 95616, USA.
Leal
Walter S
WS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D012831:Bombyx / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary; D004950:Esterases / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D009812:Odorants; D011994:Recombinant Proteins / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; D004950:Esterases