Gene amplification of the Hps locus in Glycine max

Background  

Hydrophobic protein from soybean (HPS) is an 8 kD cysteine-rich polypeptide that causes asthma in persons allergic to soybean dust. HPS is synthesized in the pod endocarp and deposited on the seed surface during development. Past evidence suggests that the protein may mediate the adherence or dehiscence of endocarp tissues during maturation and affect the lustre, or glossiness of the seed surface.     

A class I chitinase from soybean seed coat.

Protein extracts from soybean (Glycine max [L.] Merr) seed hulls were fractionated by isoelectric focusing and SDS-PAGE analysis and components identified by peptide microsequencing. An abundant 32 kDa protein possessed an N-terminal cysteine-rich hevein domain present in class I chitinases and in other chitin-binding proteins. The protein could be purified from seed coats by single step binding to a chitin bead matrix and displayed chitinase activity by an electrophoretic zymogram assay. The corresponding cDNA and genomic clones for the chitinase protein were isolated and characterized, and the expression pattern determined by RNA blot analysis. The deduced peptide sequence of 320 amino acids included an N-terminal signal peptide and conserved chitin-binding and catalytic domains interspaced by a proline hinge. An 11.3 kb EcoRI genomic fragment bearing the 2.4 kb chitinase gene was fully sequenced. The gene contained two introns and was flanked by A+T-rich tracts. Analysis by DNA blot hybridization showed that this is a single or low copy gene in the soybean genome. The chitinase is expressed late in seed development, with particularly high expression in the seed coat. Expression was also evident in the late stages of development of the pod, root, leaf, and embryo, and in tissues responding to pathogen infection. This study further illustrates the differences in protein composition of the various seed tissues and demonstrates that defence-related proteins are prevalent in the seed coat.     

Soybean seed lustre phenotype and surface protein cosegregate and map to linkage group E.

Soybean (Glycine max (L.) Merr.) seeds vary in their surface properties. The lustre, or glossiness, of seeds has been classified into several different phenotypes. Soybean seeds that have a dull lustre or moderate bloom (B) may also have abundant seed surface protein, namely, an abundance of the hydrophobic protein from soybean (HPS). The seed surface protein HPS is an allergen (Gly m 1) that causes asthma in persons allergic to soybean dust. In this study, seed lustre and surface protein content are compared among 71 different soybean cultivars and lines. Dull-seeded phenotypes usually possessed abundant surface protein in comparison to shiny-seeded types, although exceptions were observed. An F2 population of 82 individuals from a cross of OX281 (dull lustre, abundant HPS) and Mukden (shiny lustre, trace amounts of HPS) provided a basis for inheritance studies and genetic mapping analysis. Results indicate that dull seed lustre (B) and surface protein (Hps) loci are dominant Mendelian traits that cosegregate and map to soybean linkage group E. Molecular markers were used to construct a genetic map of 28 cM encompassing B and Hps. Two different molecular markers cosegregated with each of the loci. This study provides additional evidence that Hps may play a role in the adhesion of endocarp tissues to the seed, and offers new methods of selection for seed lustre and surface protein composition in soybean.     

Hydrophobic Protein Synthesized in the Pod Endocarp Adheres to the Seed Surface

Soybean (Glycine max [L.] Merr.) hydrophobic protein (HPS) is an abundant seed constituent and a potentially hazardous allergen that causes asthma in persons allergic to soybean dust. By analyzing surface extracts of soybean seeds with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino-terminal microsequencing, we determined that large amounts of HPS are deposited on the seed surface. The quantity of HPS present varies among soybean cultivars and is more prevalent on dull-seeded phenotypes. We have also isolated cDNA clones encoding HPS and determined that the preprotein is translated with a membrane-spanning signal sequence and a short hydrophilic domain. Southern analysis indicated that multiple copies of the HPS gene are present in the soybean genome, and that the HPS gene structure is polymorphic among cultivars that differ in seed coat luster. The pattern of HPS gene expression, determined by in situ hybridization and RNA analysis, shows that HPS is synthesized in the endocarp of the inner ovary wall and is deposited on the seed surface during development. This study demonstrates that a seed dust allergen is associated with the seed luster phenotype in soybean and that compositional properties of the seed surface may be altered by manipulating gene expression in the ovary wall.