EVALUATION OF GLYCINE GERMPLASM FOR NULLS OF THE IMMUNODOMINANT ALLERGEN P34/GLY M BD 30K

Authors: JOSEPH, LEINA - UNIVERSITY OF ILLINOIS; HYMOWITZ, TED - UNIVERSITY OF ILLINOIS; Schmidt, Monica; Herman, Eliot

Submitted to: Crop Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/1/2006
Publication Date: 7/1/2006
Citation: Joseph, L.M., Hymowitz, T., Schmidt, M.A., Herman, E.M. 2006. Evaluation of glycine germplasm for nulls of the immunodominant allergen P34/GLY m BD 30k. Crop Science. 46:1755-1763.

Interpretive Summary: The major human infant allergen of soybeans is a protein termed P34 or Gly m Bd 30k. Soybeans lacking this protein would provide a means to greatly enhance soybean utilization by otherwise soy-intolerant individuals. In order to attempt to develop a conventional soybean line with lower allergenicity the entire USDA soybean collection was screened to search for null varieties. Of the approximately 20,000 lines examined only two lacked P34. These two lines are null for the protein and further examination showed these two lines are either identical or very closely related. The genes and protein diversity of the P34 nulls were examined and the underlying reason for the null appears to be a mutation in the gene. The P34 null soybeans appear to otherwise normal containing all of the same proteins as the control lines. The identification and characterization of the P34 line from the USDA collection offers the prospect of developing lower allergenicity food or formula for soy-sensitive people. The results of this study should be of wide interest from industry concerned with soy allergies, the public and in particular those people who are soy-sensitive and as a trait of interest to academic and industry soy breeders who could stack this trait with other value-added traits to produce better soybeans

Technical Abstract: Soybean [Glycine max (L.) Merr.] seed contains an immunodominant human allergen, P34 or Gly m Bd 30k which belongs to the cysteine protease family. In soybean sensitive individuals, especially infants, P34 accounts for a large majority of IgE-cross reactivitity. A soybean lacking the P34 allergen (null) could improve the prospects of making soybean-formula available for sensitive individuals. The USDA soybean germplasm, containing 16,266 accessions (maintained in Urbana, IL), was screened for the presence or absence of the P34/Gly m Bd 30k protein. Twelve nulls lines were identified from the germplasm in soybean (G. max), wild annual soybean (Glycine soja Sieb. and Zucc.) and wild perennial Glycine sp. collections. Further analysis using SDS-PAGE immuno/ western blot techniques indicated that the G.soja accessions were indeed low P34/Gly m Bd 30k expressers while the G.max and wild perennial sp appeared to have null non-detectable levels of the allergenic protein. The two G. max accessions that were found to be null for the allergenic P34/Gly m Bd 30k protein were further investigated by molecular techniques. The two G. max accessions lacking P34/Gly m Bd 30k were analyzed by 2D-IEF/SDS PAGE demonstrating that all the primary seed proteins were still present indicating the loss of P34/Gly m Bd 30k is not due to a large scale restructuring of protein content. Immunoblot analysis of the 2D gels indicated that there is a very small amount of P34/Gly m Bd 30k protein present likely derived from the other nearly silenced genome of alloploid soybean. Southern analysis showed that the gene size was the same in the null P34 accessions as in the wild-type indicating that no large insertions or deletions had occurred to render the gene non-functional. Northern blot analysis from total RNA isolated from mature cotyledons showed the expected 1.1 kb transcript present in both G.max P34/Gly m Bd 30k null accessions. In order to elucidate the reason for the lack of a functional P34/Gly m Bd 30k protein in these two G.max accessions, the cDNA from both of these accessions was produced from mature cotyledons, cloned, sequenced and subsequently compared to that of the wild-type known sequence. It was found that 6 point mutations occurred in both of the cDNA sequences compared to the wildtype sequence indicating that the two null accessions are indeed a single variety. Of these six single nucleotide changes, four of them are predicted to result in an amino acid alteration. In particular one such alteration would result in a serine residue being replaced by a cysteine residue. It is reasoned that the introduction of a cysteine residue might produce a mismatched disulfide bond formation and produce an unstable P34 protein in the two null accessions identified in this study. The isolation and introgression of null soybean lines with low allergen levels will provide the basis for developing a low allergen soybean line incorporated with other agronomically desirable traits in a soybean breeding program.