Complete sequence of a cDNA of ? subunit of soybean ?-conglycinin (original) (raw)
Molecular and structural analysis of electrophoretic variants of soybean seed storage proteins
Phytochemistry, 2003
Soybean (Glycine max L.) storage proteins are composed mainly of two major components, b-conglycinin and glycinin. Electrophoretic variants of the b subunit of b-conglycinin and the A3 polypeptide of glycinin were detected on SDS-PAGE, and designated them as b* and A3*, respectively. b* and A3* exhibited higher and lower mobilities, respectively, than the common b subunit and A3 polypeptide. The N-terminal nine and 10 amino acid sequences of b* and A3* were completely identical to the previously reported sequences of the b subunit and the A3 polypeptide, respectively. Analysis using concanavalin A-horseradish peroxidase and treatment with N-glycosidase indicated that glycans were not responsible for the difference in electrophoretic mobility of b* or A3*. Furthermore, five clones of b* or b and three clones of A3*, respectively, were sequenced but we could not detect deletions and insertions except for a single or a few amino acid substitutions as compared with the common b subunit and A3 polypeptide. These results indicate that a single or a few amino acid substitution affects the electrophoretic mobilities of b* and A3*. #
Cellular localization of soybean storage protein mRNA in transformed tobacco seeds
Proceedings of the National Academy of Sciences, 1988
We transformed tobacco plants with a soybean β-conglycinin gene that encodes the 1.7-kilobase β-subunit mRNA. We showed that the β-conglycinin mRNA accumulates and decays during tobacco seed development and that β-conglycinin mRNA is undetectable in the tobacco leaf. We utilized in situ hybridization to localize β-conglycinin mRNA within the tobacco seed. β-Conglycinin mRNA is not detectable within the endosperm but is localized within specific embryonic cell types. The highest concentration of β-conglycinin mRNA is found in cotyledon storage parenchyma cells. We conclude that sequences required for embryo expression, temporal control, and cell specificity are linked to the β-conglycinin gene, and that factors regulating β-conglycinin gene expression are compartmentalized within analogous soybean and tobacco seed regions.
Plant Physiology, 1992
Protease Cl, the protease responsible for the initial degrada- tion of the a' and a subunits of the soybean ,-conglycinin storage protein (Glycine max [L.] Merrill), has been purified. The enzyme was found by sodium dodecyl sulfate-polyacrylamide gel electro- phoresis to have a molecular weight of 70,000 and a pH optimum of 3.5 to 4.5. Susceptibility to protease inhibitors indicates that protease Cl is a serine protease. Study of the proteolytic inter- mediates generated suggests that the cleavage of the a' and a subunits of 8-conglycinin by protease C1 results in intermediates that are 1 or 2 kilodaltons smaller than the native a' and a subunits. Following that, a succession of intermediates exhibiting molecular masses of 70.0 and 58.0 kilodaltons, then 63.0, 61.0, 55.0, and 53.5 kilodaltons, are observed. A 50.0-and a 48.0- kilodalton intermediate are the final products of protease Cl action. Comparison of these intermediates with the prominent anti-,8-conglycinin cross-reacting bands that increase during the first few days of germination and early growth show that protease Cl plays an important physiological role, but not an exclusive one, in the living plant. A major metabolic event in the germinating seed is the hydrolysis of the seed protein reserves to provide the growing seedling with the nutrients necessary before photosynthesis is established. There are two major storage proteins in the soybean (Glycine max [L.] Merrill) seed, glycinin and flconglycinin, which together make up 70% of the seed protein reserves by dry weight. Glycinin consists of six nonidentical subunits. Each subunit has one acidic chain and one basic chain linked by a single disulfide bond (16). ,B-Conglycinin is a glycoprotein that is composed of three noncovalently asso- ciated subunits, a', a, and # , with mol wts of 76,000, 66,000, and 47,500, respectively. During germination and early growth, these storage proteins are degraded by proteol- ysis. The predominant pattern is one of limited proteolytic cleavage by proteases specific for the reserve protein, followed by more rapid proteolysis by less specific proteases (15, 19). Several soybean proteases have been described. These include six proteolytic enzymes from ungerminated seed sep- arated by anion-exchange chromatography (26), two carbox- ypeptidases from germinating soybeans (10), two endopepti- dases, one exhibiting an acidic pH optimum and the other a
2011
The production of recombinant molecules in seeds plant has presented interesting results related to the synthesis, secretion, compartmentalisation, post-translational modi cations and puri cation, scalability, stable expression, the absence of contaminants and human pathogens, and knowledge of the growth, harvest, storage and processing practices. In this context, the soybean plant has emerged as an option to express recombinant proteins. We obtained several transgenic soybean plant lines, expressing different types of recombinant molecules, under the control of the tissue-speci c soybean seed storage βconglycinin promoter. This promoter is the most abundant protein in soybean seeds, directing the expression of the molecules that accumulate in the seed storage tissue. Cotyledonary immunocytochemical analysis of the seeds demonstrated that the targeted proteins effectively drove polypeptide accumulation into the protein storage vacuoles \(PSVs). Here, we provide a detailed protocol to reproduce the results described above and properly direct the expression of recombinant protein into the PSVs.
A Systematic Proteome Study of Seed Storage Proteins from Two Soybean Genotypes
Soybean seed is a good source of plant protein in human consumables such as baby formula and protein concentrate. The seeds contain an abundance of storage proteins, namely β-conglycin and glycinin that account for ~ 70-80% of the total seed protein content. Proteome profiling has been proved to be an efficient way that can help us to investigate the seed storage proteins. In the present study, the seeds were removed from the pods and the cotylendonary tissues were separated from the testa for proteome analysis in order to investigate the seed storage proteins. A systematic proteome profiling was conducted through one-dimensional gel electrophoresis followed by MALDI-TOF-TOF mass spectrometry in the seeds (cotyledonary tissue) of soybean genotypes. Two dimensional gels stained with CBB, a total of 10 proteins were identified and analyzed using MASCOT search engine according to the similarity of sequences with previously characterized proteins along with the UniProt database. A total of ten proteins such as glycinin Gy4 precursor, glycinin G3 precursor, glycinin G1 precursor, glycinin chain A2B1a precursor, glycinin chain A2B1a precursor were identified in our investigation. However, the glycinin subunit may be considered to play important roles in soybean breeding and biochemical characterization. In addition, the improved technique will be useful to dissect the genetic control of glycinin expression in soybean.
Plant molecular biology, 2003
The initial biochemical characterization of the soybean sucrose-binding protein, GmSBP, within our lab and others produced several incongruous characteristics that required a re-characterization of GmSBP via sequence homology, cell biology, immunolocalization, and semi-quantitative analysis. The GmSBP proteins share amino acid sequence homology as well as putative structural homology with globulin-like seed storage proteins. A comparison to the major soybean seed storage proteins, glycinin and beta-conglycinin established several storage protein-like characteristics for GmSBP. All three proteins were present in a prevacuolar compartment and protein storage vacuole. All three proteins increased in expression during seed development and are remobilized during germination. Quantitatively, the relative concentrations of GmSBP, beta-conglycinin (alpha/alpha' subunits), and glycinin (acidic subunits) indicated that GmSBP contributes 19-fold less to the stored nitrogen. The quantitativ...
Journal of Agricultural and Food Chemistry, 2007
Seed protein concentration of commercial soybean cultivars calculated on a dry weight basis ranges from approximately 37 to 42% depending on genotype and location. A concerted research effort is ongoing to further increase protein concentration. Several soybean plant introductions (PI) are known to contain greater than 50% protein. These PIs are exploited by breeders to incorporate the highprotein trait into commercial North American cultivars. Currently, limited information is available on the biochemical and genetic mechanisms that regulate high-proteins. In this study, we have carried out proteomic and molecular analysis of seed proteins of LG00-13260 and its parental high-protein lines PI 427138 and BARC-6. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis revealed that the high-protein lines accumulated increased amounts of-conglycinin and glycinins, when compared with Williams 82. High-resolution two-dimensional electrophoresis utilizing pH 4-7 and pH 6-11 ampholytes enabled improved resolution of soybean seed proteins. A total of 38 protein spots, representing the different subunits of-conglycinin and glycinin, were identified by matrixassisted laser desorption ionization time-of-flight mass spectrometry. High-protein was correlated with an increase in the accumulation of most of the subunits representing-conglycinin and glycinin. Comparisons of the amino acid profiles of high-protein soybean lines revealed that the concentration of sulfur amino acids, a reflection of protein quality, was not influenced by the protein concentration. Southern blot analysis showed the presence of genotypic variation at the DNA level between PI 427138 and BARC-6 for the genes encoding group1 glycinin,-conglycinin, Bowman-Birk inhibitor (BBI), and the Kunitz trypsin inhibitor (KTI). LG00-13260 inherited the allelic variants of the parental line PI 427138 for glycinin,-conglycinin, and KTI, while BBI was inherited from the parental line BARC-6. The results of our study indicate that high-seed protein concentration is attributed to greater accumulation of specific components of-conglycinin and glycinin subunits presumably mediated by preferential expression of these genes during seed development.