Ji-Eun Ahn
Major Professor: Dr. Keyan Zhu-Salzman
Ph.D. Candidate
"A Molecular Insight into the Transcriptional Regulation of Insect Counter Defense."
When challenged by the dietary soybean cysteine protease inhibitor, scN, the cowpea bruchid (Callosobruchus maculatus) adapts to the inhibitory effects by readjusting the transcriptome of its digestive system, including the specific activation of a cathepsin B-like cysteine protease, CmCatB. To understand the transcriptional regulation of CmCatB, we cloned the putative promoter and demonstrated its activity in Drosophila cells using a chloramphenicol acetyltransferase reporter system. Electrophoretic mobility shift assays detected differential DNA binding activity between nuclear extracts of scN-adapted and -unadapted midguts. Two tandem chicken ovalbumin upstream promoter (COUP) elements were identified in the CmCatB promoter that specifically interacted with a protein factor unique to nuclear extracts of unadapted insect guts, where CmCatB expression is repressed. Seven-up (Svp) is a COUP-TF-related transcription factor that interacted with the COUP responsive element. Polyclonal anti-mosquito Svp antibody abolished the specific DNA binding activity in cowpea bruchid midgut extracts, suggesting the protein factor is an Svp homolog. Subsequent cloning of a cowpea bruchid Svp (CmSvp) indicated that it shares a high degree of amino acid sequence similarity with COUP-TF/Svp orphan nuclear receptor family members from a variety ofspecies. The protein was more abundant in scN-unadapted insect guts than seN-adapted guts, consistent with the observed DNA binding activity. Furthermore, CmCatB expression was repressed when CmSvp was transiently expressed in Drosophila cells, most likely through COUP binding. These findings indicate that CmSvp may contribute to insect counter-defense, in part by inhibiting CmCatB expression under normal growth conditions, but releasing the inhibition when insects are challenged by dietary protease inhibitors.