plasmid probes, genes, DNA, biology, dna libraries, lepidopteran
Heliothis virescens, a member of the lepidopteran family, is of great importance to many people who rely on agriculture as a source of income because it is a pest of many important crops. Because H. virescens feeds on a broad array of hosts including cotton, tobacco, tomato, and soybean it has earned its place as a major nuisance in the United States. Heliothis subflexa is a close taxonomic relative of H. virescens, but the two species differ in that H. subflexa is a specialized herbivore that only feeds on plants in the genus Physalis. Studies have been performed to determine whether a single locus on a gene is responsible for controlling the ability of H. virescens to feed on a wide array of hosts, or whether each individual host that H. virescens feeds upon is controlled by a separate locus. By determining what locus or loci on the gene controls H. virescens’ ability to feed upon crops of agricultural importance there is a possibility to altering the gene and creating H. virescens that does not feed upon tobacco, cotton, soybean, and tomatoes. H. virescens has also been the subject of many studies regarding insecticide resistance, specifically Bacillus thuringiensis (Bt) toxin. The rise of insecticide resistance in agricultural pests is an example where a few major genes are consistently found to change in response to toxic chemical and biological agents. Bacterial Artificial Chromosome (BAC) libraries for H. virescens are important in providing a critical genomic resource to facilitate further investigations about Bt resistance. Actually identifying ecologically important genes of H. virescens requires fine scale mapping. Once the mapping has been completed, BAC libraries will be essential for isolating and sequencing candidate genes via positional cloning strategies. The purpose of this project was to evaluate the true genome coverage of the lepidopteran BAC libraries by hybridizing a set of single copy, conserved nuclear DNA sequences from H. virescens to filters from the newly constructed libraries. The BAC libraries were constructed for several lepidopteran species, including H. virescens and H. subflexa, in order to advance research into genetic mechanisms related to lepidopteran development, behavior, morphology, and evolution. In the future the isolated genes will be used by other investigators to study their structure and how they are regulated. The process for isolating probes from the plasmids began with bacterial transformation in order to introduce the DNA plasmid into bacterial cells. Next, the plasmids were isolated using a commercial kit. Once the plasmids were isolated, characterization of the plasmid was performed using agarose gel electrophoresis. This stage of the process confirmed the insert size of the plasmid was as expected. Then the plasmid was digested with restriction enzymes and purified. Finally, polymerase chain reaction (PCR) was used to amplify the inserts, which are composed of gene specific DNA, so they could be used to screen the BAC libraries.