Location: Soybean and Nitrogen Fixation Research2011 Annual Report
1a. Objectives (from AD-416)
To elucidate regulatory mechanisms of genes that control saturated fatty acid composition in sobyean seed; to characterize the mechanisms that control phytate content in seeds, and the impact of low phytate on seed viability and soy meal digestibility; to identify genes and gene products that cause immune responses to soybean and devise ways to mitigate these reactions.
1b. Approach (from AD-416)
Evaluate metabolite levels and expression of genes encoding enzymes of the phytate biosynthetic pathway of low and normal phytate seeds in response to increasing P supply; Evaluate amino acid substitution in desaturase genes; Evaluate differences in the amount of allergens bound to soy protein, determine their location in the protein, and assess variation in their structure. BSL-1. 10/01/06.
3. Progress Report
Five-prime upstream regions (500-1000 bp) of the soybean genes GmFAD2-1A, GmFAD2-1B, and GmSACPD-C, that are involved in seed oleic acid synthesis were cloned and sequenced. Thus far, by inspection, we have not found sequences indicative of promoter or regulatory features. Extending the sequencing and further sequence analysis may eventually uncover regulatory regions of these genes. Once promoter-like sequences are identified, sequences of non mid-oleic and mid-oleic soybeans will be compared in order to detect sequence polymorphisms or other features that may control the altered expression of these genes. Two Phytotron soybean day-length experiments have been completed. The purpose of these experiments was to determine the effects of two day lengths (12 hours and 9 hours) on the seed productivity and seed fatty acid composition of mid-oleic and conventional soybean varieties. The data are being analyzed and prepared for publication. A bulk seed increase of low phytate soybean lines was completed. Poultry feeding experiments with low phytate and conventional phytate soybean were begun and preliminary data are being analyzed. Inappropriate interaction of antibodies with proteins mediates the allergenic response in mammals. Previously, we developed methods that allowed the identification of soy proteins and epitopes that bind IgG but had not proved sensitive enough to identify peptides that bind the much less abundant IgEs. It is most likely that IgEs mediate the allergenic response, though IgG and IgE share a common precursor cell and are likely to have similar repertoires. By utilizing a more sensitive fluorescent ELISA, peptides that bind Dog IgE have been identified. IgE binds to more peptides than the IgG from the same individual. The fluorescent-based ELISA also provides the sensitivity to identify peptides that bind pig IgE but results are confounded by second labeled antibodies that react with pig IgG or contain antibodies that directly bind soy proteins. We are exploring alternatives to these second antibodies. The identification of regions of seed storage proteins that bind dog IgG, dog IgE, and pig IgG provide the basis to screen for soybean accessions with variants in these peptides. High resolution melting (HRM) analyses have been successful in a pilot experiment in quickly and inexpensively identifying variants of selected conglycinin genes. Forty-eight Glycine soja accessions and 380 Glycine max accessions will be screened by HRM to identify variants of sites that bind dog and pig antibodies. Available soybean mutants will also be screened. These lines will provide the genetic basis to determine if a hypoallergenic soybean line is possible. Carbohydrates associated with antinutritional factors (raffinose, stachyose and phytate) and taste components (monosaccharides, sucrose and starch) were measured in seeds of 1500 soybean mutants. Phytate was also indirectly measured by determining the level of free phosphate. To make this analytical approach affordable, a plate assay was developed that allowed the integrated screening of starch, soluble sugars and phosphate.
1. Unique day-length sensitivity of the mid-oleic mutant soybean M23. Soybean M23 has been widely employed as a breeding parent in efforts to develop a stable mid-oleic soybean. M23, produced by X-ray mutagenesis, contains a large gene disruption that deletes the GmFAD2-1A gene and at least 14 other genes. ARS researchers at Raleigh, NC discovered that M23 uniquely responds to a short day (9 hour day) by producing many pod primordia that do not develop into mature seed bearing pods, a response that was not detected in other mid-oleic soybean lines. Breeders using M23 will want to avoid the transfer of these genomic disruptions. Functional characterization of the deleted genes may provide new clues for genes involved in pod development and photoperiodicity.
2. Identification of allergenic epitopes of soybean seed storage proteins. In order to identify proteins involved in soy allergy, ARS researchers at Raleigh, NC arrayed peptides representing soybean storage proteins and developed a method to identify those that bind antibody IgE in dogs. These arrays are a robust basis for medium-throughput screening of sera to rapidly identify allergenic epitopes among seed storage proteins. Most of the regions that bind IgG also bind IgE; however, many regions only bind IgE. Repeating the experiments with live dogs will allow us to determine if binding IgE and/or IgG are associated with allergic responses. The identification of mutations of these regions will allow us to determine if hyperallergenic soybean lines are possible, and if they are possible, allow us to breed these variants into the elite soybean germplasm which will reduce soy allergy in humans.
3. Mutagenesis of soybean resulted in potentially valuable changes in seed soluble carbohydrate and starch levels. Soluble carbohydrates influence the palatability of soy meal. Carbohydrates can be antinutritional if indigestible by animals and can improve taste and calorie content if digestible. Soybeans contain the antinutritional sugars raffinose and stachyose as well as the palatable starch, sucrose and hexoses. ARS researchers at Raleigh, NC found that out of the 1500 ethyl methyl sulfonate mutagenized lines screened, 4 lines were high in starch, 4 were lines low in phytate, and 8 lines had high sucrose to raffinose ratios. The performance of these lines is being evaluated in the field this year and crosses are being made with selected lines that stably express elevated carbohydrate and/or starch and low phytate for reducing phosphorus pollution in the environment.Israel, D., Taliercio, E.W., Kwanyuen, P., Burton, J.W., Dean, L.L. 2011. Inositol metabolism and phytase activity in normal and low phytic acid soybean seed. Crop Science. 51:282-289.