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, 101 (23), 8631-6

Involvement of a Bifunctional Fatty-Acyl Desaturase in the Biosynthesis of the Silkmoth, Bombyx Mori, Sex Pheromone

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Involvement of a Bifunctional Fatty-Acyl Desaturase in the Biosynthesis of the Silkmoth, Bombyx Mori, Sex Pheromone

Ken'ichi Moto et al. Proc Natl Acad Sci U S A.

Abstract

The straight-chain C(10) to C(18) unsaturated aliphatic compounds containing an oxygenated functional group (aldehyde, alcohol, or acetate ester) derived from saturated C(16) or C(18) fatty acids are a major class of sex pheromone components produced by female moths. In the biosynthesis of these pheromone components, various combinations of limited chain-shortening and regio- and stereospecific desaturation reactions significantly contribute to the production of a vast number of the species-specific pheromone components in Lepidoptera. Biosynthesis of the silkmoth sex pheromone bombykol, (E,Z)-10,12-hexadecadien-1-ol, involves two consecutive desaturation steps, the second of which is unique in that it generates a conjugated diene system from the Delta11-monoene C(16) intermediate. In experiments designed to characterize the acyl-CoA desaturases responsible for bombykol biosynthesis, we have cloned three cDNAs encoding desaturase family members from the pheromone gland of the inbred strain of the silkmoth, Bombyx mori. Transcript analyses by RT-PCR and subsequent functional assays using a Bac-to-Bac baculovirus expression system revealed that desat1 is the only desaturase gene prominently expressed during pheromonogenesis and that its gene product, B. mori Desat1, possesses both Z11 desaturation and Delta10,12-desaturation activities. Consequently, we have concluded that B. mori Desat1 is not only a bifunctional desaturase involved in bombykol biosynthesis but that it is also the enzyme responsible for both desaturation steps.

Figures

Fig. 1.
Fig. 1.
Fractionation of the 566-bp PCR products containing pheromone gland desaturase gene fragments after sequential digestions with restriction enzymes. Lane 1, undigested PCR products; lane 2, XhoI digests separating desat1 fragments; lane 3, ScaI digests after XhoI digestion; M, size markers.
Fig. 2.
Fig. 2.
Multiple alignment of B. mori pheromone gland fatty-acyl desaturases. The consensus residues are highlighted in black, and the residues that exhibit similarity are shaded in gray. The three conserved histidine clusters are overlined. The regions corresponding to the primer sequence used for RT-PCR are indicated by arrows. Alignment was made by using the clustalw program (http://hypernig.nig.ac.jp/homology/clustalw.shtml) and boxshade 3.21 file (www.ch.embnet.org/software/BOX_form.html).
Fig. 3.
Fig. 3.
RT-PCR analysis of desat transcripts from B. mori tissues. Tissues were dissected from newly emerged female moths (day 0). Testes were dissected from newly emerged male moths. 1, pheromone gland; 2, head; 3, flight muscle; 4, Malpighian tubule; 5, testis; 6, ovary; M, size markers.
Fig. 4.
Fig. 4.
GC/MS analysis by total ion current for the FAMEs prepared from Sf9 cells infected with the recombinant Desat1-AcNPV or the recombinant Bac1-AcNPV (control). (A) Sf9 cells infected with Bac1-AcNPV. (B) Sf9 cells infected with Desat1-AcNPV. (C) Sf9 cells infected with Bac1-AcNPV and incubated in the presence of Z11–16:acid.
Fig. 5.
Fig. 5.
GC/MS analysis by total ion current or mass chromatograms for the FAMEs prepared from Sf9 cells. Sf9 cells were infected with Bac1-AcNPV (A–C) or Desat1-AcNPV (D–F) and incubated in the presence of 0.1 mM D3-C16:acid; m/z 269, [M]+ of D3-diene C16:Me; m/z 271, [M]+ of D3-monoene C16:Me; m/z 273, [M]+ of D3-C16:Me; m/z 297, [M]+ of D3-diene C18:Me; m/z 299, [M]+ of D3-monoene C18:Me; m/z 301, [M]+ of D3-C18:Me.
Fig. 6.
Fig. 6.
Phylogeny of various lepidopteran desaturases. Tree was reconstructed by using the phylogeny inference package (phylip) by Felsenstein (21) (http://bioweb.pasteur.fr/seqanal/phylogeny/phylip-uk.html). The accession numbers for the sequences are listed to the right of the species abbreviation. The B. mori desaturase genes are listed in bold.

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