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:
Progress was made in the determination and comparison of the proteins present in the seed of a group of 27 different soybean lines. These included elite soybean varieties, a diverse group of unimproved soybean (Glycine max) lines and G. soja (wild soybean) germplasm accessions from the USDA Soybean Germplasm Collection. To study the natural seed protein variation among these lines, we used a “proteomics” approach in which seed proteins were separated, identified, and quantified using mass spectrometry. We separated the seed proteins using a procedure called 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The resulting protein spots on the gels were scanned and compared among different soybean accessions and the quantity of protein in each spot was assessed using image analysis software. Different types of proteins including storage, allergen, and anti-nutritional proteins were identified using integrated mass spectrometry (Matrix-assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry and Liquid Chromatography Mass Spectrometry). We found major and minor variations in the amount and number of protein subunits between wild and cultivated soybean lines. Similar analyses were conducted to determine the variation of protein profiles between transgenic (also referred to as genetically modified (GM)) and non-transgenic soybean seeds. A database was developed that incorporates these protein data. A detailed analysis of these protein profiles is in progress. Progress was made in the comparison of three methods of protein extraction to find the most suitable method for protein extraction from soybean root and soybean cyst nematode tissues. Each extraction was followed by mass spectrometry in order to identify the extracted proteins. Of the 3 methods, the “phenol extraction method” was the most productive, as it allowed us to match 816 proteins, with 426 of them being unique proteins. The Gene Ontology database which describes the function of genes was used to assign a function to each of these proteins. Our results showed that proteins involved in metabolic, developmental, and biological regulation processes were the most abundant.
1. Characterization of cyst nematode proteins. The soybean cyst nematode (SCN) is the major pest of soybean in the U.S. To improve soybean yields by increasing the level of plant resistance to this important soybean pest, it is important to understand the protein composition of the nematode. Therefore, the ARS researchers at Beltsville, MD have evaluated, standardized and applied the technology to determine and quantify the spectrum of proteins present in the soybean cyst nematode. To do this, the researchers are using a “proteomics” approach in which nematode proteins are separated, identified and quantified using a device called a mass spectrometer. They evaluated an efficient extraction method to identify more nematode proteins. This approach will be important in providing a way to identify nematode proteins and to develop a reference map of nematode proteins This standardized methodology for defining and quantifying the spectrum of protein in nematode will be useful to scientists who wish to develop nematode resistant soybeans in order to improve overall soybean yields.
Chen, X., Macdonald, M.H., Garrett, W.M., Matthews, B.F., Natarajan, S.S. 2011. Evaluation of protein extraction methods suitable for two-dimensional gel electrophoresis of the soybean cyst nematode (Heterodera glycines). Analytical Biochemistry. 41(2):240-247.