(moths. are produced de novo in the pheromone gland (PG) of

(moths. are produced de novo in the pheromone gland (PG) of woman moths from a common fatty acid, palmitic acid, through several enzymatic reactions (i.e., limited oxidation, desaturation, reduction, and acetylation) (11C14). A desaturase specifically indicated in the PG is the key enzyme that introduces a double relationship into the pheromone molecules PP121 (15C19). A single 11-desaturase of isomer (9). In the present study, through the cloning and practical analysis of a PG-specific 11-desaturase in isomer in the pheromone is definitely attributable to the rigid product specificity of the 11-desaturase with this varieties, LATPG1. We also display that LATPG1 is definitely closely related to retroposon-linked cryptic 11-desaturases (and (21). The development of 11-desaturases in is definitely discussed in light of these findings. Results Pheromone Precursors in the PG of To elucidate the crucial biosynthetic step that determines the absence of the PP121 isomer in the pheromone of percentage of pheromone precursor acids in the PG of isomer of the pheromone precursor was present in the PG (Fig. 1), suggesting the 11-desaturase with this varieties produces only E11-14:Acyl. Fig. 1. Fatty acid analysis of PG components from by GC-MS. Total ion chromatograms of methyl ester derivatives of fatty acids in the PG (solid collection), and of authentic standards (broken collection), ( To identify the 11-desaturase that generates only the isomer, we performed PCR using degenerate primers designed based on the histidine-cluster sequences conserved in nonheme desaturases (16, 17). We cloned and sequenced 570-bp cDNA fragments, and found that 44 of 47 clones showed the same sequence, which was different from those of 11-desaturase genes recognized from your congeners. We named this unique gene (pheromone gland-specific desaturase 1). The remaining three clones were fragments of 9-desaturase or ubiquitous genes, such as 16S rRNA. The full-length sequence of was acquired by 3- and 5-RACE experiments. The gene comprised a 957-bp ORF encoding a protein of 319 amino acid residues having a expected molecular mass of 37.8 kDa. Sequence analysis exposed that LATPG1 offers features conserved in insect nonheme desaturases: four transmembrane domains, three histidine boxes, and a signature motif (Fig. 2). The amino acid sequence of LATPG1 showed 55.7 PP121 to 56.7% identity to 11-desaturases of congeners, (Fig. 2). Fig. 2. Assessment of deduced amino acid sequences of LATPG1 and 11 desaturases from three additional varieties (in several cells of by RT-PCR. The mRNA was specifically indicated in the PG (Fig. 3), suggesting that LATPG1 is the major desaturase involved in pheromone synthesis in analyzed by RT-PCR. Cells distribution of mRNA. RT-PCR analysis was performed using cDNA prepared from different cells of adult virgin females (1- to 3-d-old). The actin gene was used like a control. FM, flight muscle mass; … In Vitro Functional Assay of LATPG1. To verify the enzymatic activity of LATPG1, we generated recombinant baculoviruses expressing LATPG1 (LATPG1-AcNPV) or His-tagged LATPG1 (LATPG1-His-AcNPV). Western blot analysis of proteins extracted from Sf9 cells infected with LATPG1-His-AcNPV shown that a His-tagged LATPG1 having a molecular mass of 37 kDa was indicated (Fig. 4geometry at position 11 of tetradecanoic acid. Given that only the isomer of the pheromone precursor was found in the PG, we conclude that generates its pheromone using an unusual 11-desaturase, LATPG1. Fig. 4. Enzymatic activity of LATPG1 protein indicated by recombinant baculoviruses. (isomer (16). Fig. 5. Phylogenetic analysis of LATPG1 along with other desaturases. (was used as an out-group. Bootstrap ideals after 1,000 replications … A BLAST search of general public databases exposed that LATPG1 is definitely closely related to retroposon-linked cryptic 11-desaturases (and (Fig. 5and homolog in the PGs of additional varieties by RT-PCR. The manifestation of homolog was not observed in additional congeners examined (Fig. MCM2 6homolog was consistent with the presence/absence of Z11-isomer in the pheromone; that is, homolog was observed in varieties with Z11-isomer (and sex pheromones. (and genes in the PG of five varieties. (and contains five retroposon-linked cryptic 11-desaturase genes (ECB was inferred by Xue et al. (21). After the duplication of an ancestral 11-desaturase gene, a Collection retroposon named the element was put into one of the copies. The copy without the retroposon developed into the varieties, and and isomers may be PP121 a derivative characteristic. This mutation might have contributed to the.

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