Isolation and characterization of soybean vegetative storage proteins and genes
Abstract
Soybean vegetative storage proteins (VSPs) were purified from soybean leaves and characterized. Reversed-phase HPLC resolved a partially purified mixture of VSPs into three fractions. One fraction contained only 27 kD VSP, and the other two contained 29 kD VSP. Analyses of these proteins indicated that the two 29 kD VSPs had very similar amino acid sequences, and that the 27 kD VSP was homologous to the 29 kD VSPs. Previous data indicated that the VSPs were isolated from leaves as dimers (Wittenbach, 1983b). To investigate further the nature of the VSP subunit interactions, the partially purified VSP mixture described above was resolved into several fractions by anion exchange HPLC. The order of elution and content of the fractions, together with cross-linking experiments, indicated that one fraction contained 27 kD/27 kD homodimers, one 29 kD/29 kD homodimers and one 27 kD/29 kD heterodimers. Two genomic clones that contained 29 kD VSP coding sequences were isolated and characterized. One clone contained a complete 29 kD VSP gene. Nucleotide sequence analysis of the gene and its flanking regions showed that the gene was composed of three exons and two introns. Putative CAAT and TATA boxes, and regions with homology to an animal enhancer core sequence, were identified in the 5$\sp\prime$ flanking region. A putative polyadenylation signal was identified in the 3$\sp\prime$ flanking region. The second clone contained the 5$\sp\prime$ regions of two partial 29 kD VSP genes in an inverted linkage. Genomic DNA gel blot analyses showed that this arrangement represented the organization of the 29 kD VSP genes in the soybean genome. A chimeric gene was constructed that consisted of the immediate 1.5 kb upstream flanking region of the 29 kD VSP gene fused to a $\beta$-glucuronidase (GUS) reporter gene. The chimeric gene was transferred into tobacco using Agrobacterium tumefaciens, and plants were regenerated. These studies have extended our knowledge about the structure of the soybean vegetative storage proteins and the genes that encode them. This information, and the genomic clones that were isolated, should be useful in designing experiments to further an understanding of how VSP gene expression is regulated, and the role of VSPs in plant development.
Degree
Ph.D.
Advisors
Nielsen, Purdue University.
Subject Area
Botany|Molecular biology|Agronomy
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