Evaluation of Glycine Germplasm for Nulls of the Immunodominant Allergen P34/Gly m Bd 30k

Abstract

Soybean [Glycine max (L.) Merr.] seed contains an immunodominant human allergen P34 or Gly m Bd 30k (mentioned as P34) of the cysteine protease family. of approximately 16 266 accessions from USDA soybean germplasm screened, 12 P34 null lines were identified among soybean (G. max), wild annual (Glycine soja Sieb. and Zucc.), and wild perennial Glycine spp. Glycine soja were low P34 expressers, while G. max and wild perennial species had nondetectable levels of the allergenic protein. Further investigation of G. max nulls by 2D‐IEF/SDS PAGE showed all primary seed proteins present indicating that the loss of P34 was not due to large scale restructuring of protein content. Southern and northern analysis showed no large insertions or deletions to render the gene nonfunctional. The cDNA of both G. max nulls each showed the same six point mutations indicating the two nulls have a single origin. of these six single nucleotide changes, four are predicted to result in an amino acid alteration. One such alteration results in a serine being replaced by a cysteine residue. The introduction of a cysteine residue might produce a mismatched disulfide bond formation producing an unstable P34 protein in the null soybean accessions. The isolation and introgression of soybean lines with low allergen levels will provide the basis for developing a low allergen line incorporated with other agronomically desirable traits in a breeding program.

Soybean Allergens Affecting North American Patients Identified by 2D Gels and Mass Spectrometry

“…These findings seem to cast doubt on the previous reports that the soybean P34/Gly mBd 30K allergen is the most immunodominant allergen in the population (Ogawa et al 1991;Joseph et al 2006) since several other proteins are as significant, if not more significant, in this cohort. Some early reports of soy allergen identification by Western blot have used sera from children (Ogawa et al 1991); however, our study has demonstrated that, although this protein was frequently detected by adult IgE, it was not responsible for intense reactions (see band 11 on Fig.…”

“…In fact, preliminary results of patient serum screening on 2D gels would seem to indicate that these two allergens are not major or are not being detected by 2D gel screening (data not shown). We are presently making use of a recently discovered P34-null germplasm to further investigate these North American patients (Joseph et al 2006). Although this group of patients is relatively small in number, it suggests a trend with regard to the soybean proteins most likely to provoke allergic reactions among North Americans.…”

Inactivation of allergens and toxins

“…Comparing the techniques adopted for reducing toxins and allergens, usually transgenesis shows superior characteristics: it is not only more efficient in obtaining the desired phenotype (both in time and trial numbers) but also more precise. Natural null mutants for the P34 soybean allergen [117] have a frequency of 2/14,000 in cultivated soybean, that is 0.014%, and the exact reason for the lack of P34 is uncertain. Conversely, the frequency of soybeans coming out of a transformation showing P34 cosuppression is in the 10-20% range (E. Herman, pers.…”

“…Therefore an alternative approach was used to achieve the same goal: identify soybeans lacking the allergen. The entire USDA national soybean germplasm collection was screened and out of more than 16,000 accessions screened, they found 12 lines (2 of which are cultivated soybean) with no P34 allergen [117]. Based on the sequence analysis, it is possible to guess the reason why these soybean plants lack the allergen.…”

Development of Low Gly m Bd 30K (P34) Allergen Breeding Lines Using Molecular Marker in Soybean [Glycine max (L.) Merr.]

“…Of the approximately 16,266 soybean accessions from the USDA germplasm collection that were screened, only two were found to have reduced P34 protein accumulation in seeds (Joseph et al 2006). The low P34 soybean accessions PI 567476 and PI 603570A were characterized as having normal levels of seed proteins other than P34 (Joseph et al 2006).…”