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ARS Home » Midwest Area » Columbia, Missouri » Plant Genetics Research » Research » Publications at this Location » Publication #318415

Research Project: Modification of Soybeans for Food, Feed, and Industrial Applications

Location: Plant Genetics Research

Title: Analysis of soybean tissue culture protein dynamics using difference gel electrophoresis

Author
item Miernyk, Jan
item Jett, Alissa - University Of Missouri
item Johnston, Mark

Submitted to: Journal of Proteomics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/26/2015
Publication Date: 9/3/2015
Publication URL: http://handle.nal.usda.gov/10113/61666
Citation: Miernyk, J.A., Jett, A., Johnston, M.L. 2015. Analysis of soybean tissue culture protein dynamics using difference gel electrophoresis. Journal of Proteomics. 130:56-64.

Interpretive Summary: A multitude of technical problems interfere with the consistent staging of developing soybeans for systems-type analysis. An immortalized cell line suitable for growth in liquid suspension was developed from immature embryonic axes. The suspension cultures were envisioned as a readily available uniform source of cells for biochemical and molecular analyses. Both cellular and extracellular proteins were analyzed at regular stages during a growth sequence. Unexpectedly both cellular and extracellular proteins displayed a dynamic pattern of abundance. The suspension cultures are a potential resource for studying specific aspects of soybean development, but are not suitable as an alternative to study of terminally differentiated seed cells, tissues, and organs. The information will be most useful to other scientists, including breeders and those using a biotech-approach to modifying seed composition.

Technical Abstract: Excised hypocotyls from developing soybean (Glycine max (L.) merr. cv. Jack) were cultivated on agar-solidified medium until callus formed. The calli were then propagated in liquid medium until stable, relatively uniform, finely-divided suspension cultures were obtained. Cells were typically transferred to fresh medium at 7-day intervals. Cultures were harvested by filtration five days (early log phase) or eight days (late log phase) after transfer. In order to evaluate dynamic changes, both intracellular and extracellular proteins were analyzed by 2-dimensional difference gel electrophoresis. Selected spots were subjected to in gel tryptic-digestion and the resultant peptides analyzed by nLC-MS/MS. In follow-up studies gel-free shot-gun analyses led to identification of 367 intracellular proteins and 188 extracellular proteins.