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

^a Dep. of Crop Sciences, Univ. of Illinois, Turner Hall, 1102 S. Goodwin Ave., Urbana, IL 61801
^b USDA-ARS, Donald Danforth Plant Science Center, 975 N. Warson Rd., St. Louis, MO 63132

View larger version (65K): [in a new window]   Fig. 1. One-dimensional protein profile and western blot analysis of P34-low expressing accessions. Total protein was extracted from mature seeds, separated on a 4 to 18% (w/v) gradient gel, stained with Coomassie blue [in each case upper figure (i)], and a replicate gel blotted for immuno-detection using the P34 specific monoclonal antibody. Control cv. Kunitz is compared with seven accessions of G. soja (A) three wild perennials, * IL = University of Illinois no. IL0827 (PI509462), IL 0612 (PI 446991), IL 0956 (PI not known) (B) and two G. max accessions (C).

View larger version (81K): [in a new window]   Fig. 2. Proteome analysis of two G. max P34-null accessions by 2-dimensional protein analysis. Proteins were separated using a pH gradient from 3.0 to 10.0 in the first dimension and 4 to 16% (w/v) gels in the second. Total proteins were detected by Coomassie blue stain [in each case, upper gel (i)] and a western blot performed on a replicate gel using P34-specific monoclonal antibody [in each case lower blot (ii)]. The cv. Kunitz (A) was used as a positive control and compared with the two P34-null accessions G. max PI603570A (B) and G. max PI567476 (C).

View larger version (68K): [in a new window]   Fig. 3. Southern blot analysis of total DNA from G. max P34 null accessions. Samples were digested with either HaeIII or EcoRI and probed with a 32P-labeled 1.1-kb P34 amplicon. Banding pattern was compared with that of a control cv. Jack

View larger version (39K): [in a new window]   Fig. 4. Northern blot analysis of G. max P34 null accessions. Total RNA was isolated from mature cotyledons and probed with a 32P-labeled 1.1-kb P34 amplicon. Upper panel is the ethidium bromide stained gel for a loading control. The cv. Jack was used as a control.

View larger version (72K): [in a new window]   Fig. 5. Comparison of predicted amino acid sequences of the seed allergen P34 from two G. max P34 null accesssions. The pre-determined pre-pro peptide sequence is underlined. Translation of the P34 cDNA obtained from GenBank accession J05560. * denotes an alteration in the amino acid sequence.