Poster: Regulation of gene expression

Abs # 776: Subtilisin-related proteases in arabidopsis thaliana seedlings

Presenter:	Li, Ming , ming.li@binghamton.edu
Authors	Li, Ming  (A)   Wilson, Karl A (A)   Tan-Wilson, Anna  (A)
Affiliations:	(A): State University of NY at Binghamton

Arabidopsis thaliana has 56 subtilisin-related protease (subtilase) genes, but it is unknown what role these proteases might play. Based on the RT-PCR results on day-7 seedlings of Arabidopsis thaliana, 29 subtilases are expressed at mRNA level. Some are expressed at higher levels than others. There are also differences in gene expression when seedlings are etiolated as compared to the expression when seedlings are grown in a 16 h photoperiod. Determining which of these difference levels translate to the protein level is hampered by the size of this multi-gene family. It is not cost-effective to produce antibodies that bind exclusively to each subtilase. Instead, we produced two antibodies against peptides bearing conserved amino acid sequences on these subtilases, the goal being to isolate or detect the subtilases and identify them by mass spectrometric methods. To check out the abilities of the antibodies to bind to different subtilases, cDNA clones were expressed in E. coli, and checked by Western blot for binding to the antibodies. Eleven of 14 products made from full-length or partial cDNA clones were recognized by at least one of the antibodies. Western blot of a 2D-PAGE of proteins from seedling leaves showed at least three spots representing proteins recognized by the antibodies. The next steps involve fractionation of plant proteins by a combination of ion-exchange, affinity chromatography and 2D-PAGE, followed by identification by peptide mass fingerprinting and some sequencing by post-source dissociation.