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Date of Award
Bachelor of Science (BS)
Department of Biology
Jonathan D. Monroe
BAM2 is a chloroplast-targeted member of the β-amylase gene family that currently has an unknown function in starch hydrolysis. Previous research indicated that BAM2 did not have significant catalytic activity, but, because the gene is highly conserved and there is a starch-excess phenotype in older plants lacking BAM2, it was hypothesized that BAM2 may instead have a regulatory function. Many regulatory proteins function by interacting with other proteins, so we wanted to test for potential protein binding partners for BAM2 using the yeast two-hybrid system. A plasmid containing the BAM2 gene from Arabidopsis thaliana was co-transformed into Y190-strain yeast (Saccharomyces cerevisiae) along with a second plasmid from a library of plasmids containing cDNA of mRNA isolated from mature Arabidopsis leaves and roots. If BAM2 interacts with any of the proteins encoded within the cDNA library, transcription in that co-transformed yeast is activated for certain reporter genes whose expression is monitored by growth on selective media and a colorimetric assay. We observed 23 colonies with expression of both reporter genes from the first cDNA library screen and 55 colonies from a second screen. Plasmids containing cDNA were extracted from these colonies and are being tested further for false positives. The cDNA insert in plasmids from 45 potential positive results were sequenced and analyzed to provide information about the validity of interaction with BAM2. One putative positive was identified as an uncharacterized chloroplastic protein, which, after further validation, may represent a previously unidentified protein involved in starch metabolism and may reveal the function of BAM2 in the process of starch hydrolysis.
Hines, Megan E., "Searching for potential binding partners of Arabidopsis β-amylase2 using yeast 2-hybridization" (2016). Senior Honors Projects, 2010-current. 210.