1a. Objectives (from AD-416)
The overall aim of this research project is to evaluate the quality and safety of transgenic soybeans using proteomics technologies. We will determine whether the expression of transgenes in soybeans results in an unusual accumulation of non-target proteins in the seed. The data from these studies would be useful to measure and compare the deviation of protein variation within transgenic soybeans. To achieve this goal, we propose the following two key objectives: Objective 1. Measure the natural variation of seed proteins in a wide range of soybean germplasm using proteomics and establish a baseline for subsequent estimation of collateral effects that may be associated with transgenes. Objective 2. Determine the potential collateral effects in protein expression resulting from tissue culture, transformation protocols, and transgenic events in soybean.
1b. Approach (from AD-416)
The experimental approach is to compare and characterize protein variation of a group of twenty-seven soybean genotypes that will include wild, Glycine soja and cultivated Glycine max germplasm accessions. Also, variation in seed protein levels in four different seed samples from plants derived via tissue culture and non-tissue culture methods will be compared. Proteins will be extracted from seeds using the modified TCA/acetone method and separated using 2D-PAGE, analyzed using image analysis and in those cases when unique protein spots appear in any treatment, further characterization using MALDI-TOF-MS will be undertaken.
3. Progress Report
The proteins present in the seed of a group of 27 soybean genotypes were compared and characterized. These included elite soybean varieties and a diverse group of unimproved Glycine max (cultivated soybean) and G. soja (wild soybean) germplasm accessions. The 27 soybean genotypes consist of genotypes of three different Maturity Groups (early, medium and late). Each Maturity Group was represented by nine different genotypes and was planted in Brookings, SD; Beltsville, MD; and Clemson, SC. To study the natural seed protein variation, we used a proteomics approach in which seed proteins were separated, identified and quantified using mass spectrometry. Multiple seed protein extractions were performed on each replicate seed sample to accurately assess the variation associated with protein extraction and the analytical procedures we employed. Different types of proteins including storage, allergen and anti-nutritional proteins were identified. For the various seed proteins, we found both major and minor variation in the amount and number of protein subunits between wild and cultivated soybean genotypes. In addition, we characterized the protein variation between transgenic and non-transgenic soybeans.
1. Natural variation of soybean seed proteins using proteomics. Soybean is the second most valuable crop in the U.S. with an estimated annual value of ~$31.7 billion. Genetically modified (GM) soybean is widely grown in the U.S. Therefore, it is important to determine if any unintended changes occur in the soybean seed as a result of the genetic modification. To understand the effect of GM on protein variation, it is important to determine the natural variation of protein composition of wild and cultivated soybeans that have been, or may be used in conventional soybean breeding programs. These data on natural variation of seed proteins can then be compared with the variation in GM soybean to determine if the variation in GM soybean falls outside of the limits of natural variation. Data on natural variation in soybean seed protein composition were generated by assessing a diverse set of wild and cultivated non-transgenic soybeans grown in diverse environments. These seed protein data, collected on genetically diverse soybeans grown in diverse environments, clearly define the extent of variation that can be anticipated in normal non-GM soybean and provide the basis for scientists to determine whether the levels of different proteins in GM soybeans are within the limits of natural variation.
Natarajan, S.S., Xu, C., Cregan, P.B., Caperna, T.J., Garrett, W.M., Luthria, D.L. 2008. Utility of proteomics techniques for assessing protein expression. Journal of Regulatory Toxicology and Pharmacology. 54:S32-S36.