Progress 09/15/02 to 09/14/05
Outputs
This project was focused on pheromone desaturase genes in the European corn borer(ECB) moth. Research with genomic DNA has been carried out to provide the basis for a large evolutionary genomics study combined with functional biochemical analyses with various selected species to provide glimpses into the genomic architecture of moths and insights into the organization and diversification of sex-pheromone desaturase genes. The research was initiated with genomic DNA of ECB by construction of a GenomeWalker genomic DNA library. Screening of these libraries has provided some intriguing results. First, we found stretches of sequence in the ECB-D11 promoter region that are identical to upstream regions in the mammalian trypsin and hJHBP genes. Although the significance of this homology has yet to be determined, we hypothesize that these regions of similarity correspond to regulatory element binding sites; this hypothesis, of course, requires experimental testing and
confirmation. Second, we found three variants of the D11 desaturase gene in both ECB and ACB genomes. Our previous cDNA studies revealed the presence of only one D11 gene. When we compared the ECB-D11 variants with the ACB-D11 variants, we found that they all have the same exon-intron pattern, but they differ with respect to their intron lengths. Intriguingly, all of the variants are flanked by retroposons. The promoter regions of the ECBv1 and ACBv1 gene contain a reverse transcriptase coding sequence that we found to be a member of the RTE-1 family of long-interspersed elements (LINEs). It is most closely related to a LINE from C. elegans, and is noticeably absent in both the silkworm and fruitfly genomes. We also found that exon 1 and intron1 of ECBv2, ACBv2, ECBv3 and ACBv3 have been precisely removed and replaced with an unusual type of retroposon. The sequence from this element lacks homology to any deposits (including complete genomes) in GenBank, but it is most similar to
members of the non-LTR group of retroposons. Five clones of this element have been isolated and found to contain an ORF with similarity to reverse transcriptases (rtORF), but the upstream sequences are highly divergent from its closest matches in GenBank. Retroposons could provide a mechanism explaining the multiplication of the D11 gene in the ECB and ACB genomes. We also characterized the genomic sequence of the D14 desaturase gene that is not expressed in ECB, but is the active pheromone desaturase in the related Asian corn borer. It contains 4 introns in positions similar to those found in mouse and Drosophila Z9-desaturase genes, but contains a protein translation initiation factor in its promoter region. As with the D11 variants, we have not yet conducted functional assays on this sequence. Research also was initiated with an ECB bacterial artificial chromosome (BAC) library. This library has an average insert size of 125 kb and provides 9X coverage of the ECB genome. To date we
have pulled out 37 positive clones using mixed probes that target the D14 and D11 genes, as well as several D11 variants. Sequencing the clones has already confirmed that we have a D11 gene and two clones for D11 Variant2 genes.
Impacts
The research provides an insight into how corn borer species have evolved relative to changes in their sex-pheromone communication systems. The studies specifically elucidate the evolutionary history of pheromone desaturases and provide information on the mechanisms by which they have diversified and formed a multigene family. Research initiated with the BAC libraries will help determine if the desaturase genes are clustered, how many clusters exist, and how both genes and clusters are organized across large segments of the ECB genome in relation to other genes and genetic elements.
Publications
- Liu, W., Rooney, A. P., Xue, B., Roelofs, W. L. 2004. Desaturases form the spotted fireworm moth (Choristoneura parallela) shed light on the evolutionary origins of novel moth sex pheromone desaturases. Gene 342: 303-311.
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Progress 01/01/04 to 12/31/04
Outputs
This project is focused on pheromone desaturase genes in the European corn borer(ECB) moth. Research with genomic DNA has been carried out to provide the basis for a large evolutionary genomics study combined with functional biochemical analyses with various selected species to provide glimpses into the genomic architecture of moths and insights into the organization and diversification of sex-pheromone desaturase genes. The research was initiated with genomic DNA of ECB by construction of a GenomeWalker genomic DNA library. Screening of the GenomeWalker genomic DNA library from ECB revealed the presence of 3 variants of the D11 desaturase gene, which had been previously characterized to produce a mixture of Z and E11-14:Acid products as pheromone precursors. However, previous cDNA studies have revealed the presence of only 1 gene. One D11 variant matches the known desaturase gene of ECB-Z/E11. Another D11 variant contains stretches of sequence that are homologous to
trypsin and hJHBP genes in its promoter region. Its full-length cDNA sequence containing the conceptual ORF has been isolated from the ECB pheromone gland cDNA library. These two have about 70 percent amino-acid identity, with the same exon-intron pattern but differ in intron length. Although the functionality of the latter has not yet been determined, their variances indicate they are distinct genes. Finally, a third variant was found to contain a reverse transcriptase coding sequence. In BLAST searches, we determined that this coding sequence was homologous to a reverse transcriptase found in a C. elegans transposon. Although its full-length cDNA has not been isolated, we note that transposable elements have the capacity to alter transcription. As such, it might have contributed to the alteration of the ECB pheromone blend that preceded speciation from ACB by altering the expression pattern. We also characterized the genomic sequence of the D14 desaturase gene that is not expressed
in ECB, but is the active pheromone desaturase in the related Asian corn borer. It contains 4 introns in positions similar to those found in mouse and Drosophila Z9-desaturase genes, but contains a protein translation initiation factor in its promoter region. As with the D11 variants, we have not yet conducted functional assays on this sequence.
Impacts
Data from this research will provide an insight into how chemical communication systems can evolve by the switching on of nonfunctional genes. It could help explain the conundrum of how stabilized pheromone systems can rapidly change to different pheromone components.
Publications
- Liu, W., Rooney, A. P., Xue, B., Roelofs, W. L. 2004. Desaturases form the spotted fireworm moth (Choristoneura parallela) shed light on the evolutionary origins of novel moth sex pheromone desaturases. Gene 342: 303-311.
- Rodriguez, S., Hao, G., Liu, W., Pina, B. Rooney, A., Camps, F., Roelofs, W., Fabrias, G. 2004. Expression and evolution of delta-9 and delta-11 desaturase genes in the moth Spodooptera littoralis. Insect Biochem. Molec. Biol. 34: 1315-1328.
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Progress 01/01/03 to 12/31/03
Outputs
Research was conducted on the delta-11 and delta-14 desaturase genes that were characterized in both the European corn borers (ECB), Ostrinia nubilalis, and the Asian corn borer (ACB), O. furnacalis. One goal was to determine the desaturase-gene expression levels in these two species. The expression levels of the four desaturase genes (includes two delta-9 genes) found in both the pheromone gland and fat body of these two species were determined by real-time PCR. In the ECB gland the delta-11 gene was found to be the most abundant, although there was a significant amount of the delta-14 gene. There was no significant difference in the expression level of the two delta-9 genes and the delta-14 in the fat body, but the delta-11 gene was the most abundant. In the ACB gland the delta-14 gene was the most abundant and its expression level was found to be more than 100 times higher than the other three. In the fat body the delta-11 gene was again found to be the most
abundant. Another aim is to isolate and analyze full-length desaturase genes in corn borers. Based on our present information, there are two introns in the delta-11 gene, one is 167 bp, another one is 929bp; and there are three introns in the delta-14 gene, which are are 562bp, 534 bp and 602bp. A third aim is to isolate the promoter region of the desaturase genes. A GenomeWalker kit was used to isolate the promoter region of the known genes. The fragments of about 500 bp upstream of the known start site of the delta-11 and delta-14 genes were isolated and sequenced. Longer fragments upstream of the start site will be isolated and analyzed. A fourth aim is to conduct Western blotting studies to determine if the desaturase proteins are produced in both the ACB and ECB species. After many failed trials, good antibodies specific to the desaturases were generated by injecting 13-15 aa synthetic peptides into New Zealand White rabbits. They are with high titer and specific to the
desaturases with no interaction among the desaturases. This work is still continuing.
Impacts
Data from this research will provide an insight into how chemical communication systems can evolve by the switching on of nonfunctional genes. It could help explain the conundrum of how stabilized pheromone systems can rapidly change to different pheromone components.
Publications
- No publications reported this period
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Progress 01/01/02 to 12/31/02
Outputs
No Progress - Just Initiated.
Impacts
(N/A)
Publications
- No publications reported this period
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