Isolation and Characterization of Messenger RNAs for Seed Lectin and Kunitz Trypsin Inhibitor in Soybeans

Abstract

The mRNA for seed lectin and Kunitz trypsin inhibitor of soybean were highly enriched by immunoadsorption of the polysomes synthesizing these proteins. Polysomes isolated from developing seed of cv. Williams were incubated with monospecific rabbit antibodies produced against lectin subunits or trypsin inhibitor protein. The polysomal mixture was passed over a column containing goat anti-rabbit antibodies bound to Sepharose. Bound polysomes were eluted and the mRNA was selected by passage over oligo-cellulose. Lectin complementary[c]DNA hybridized to an 1150-nucleotide message and trypsin inhibitor cDNA hybridized to a 770-nucleotide message in blotting experiments using total poly(A)RNA. Translation of soybean lectin mRNA using a rabbit reticulocyte lysate yielded a major polypeptide of 32,300 whereas the MW for purified lectin subunits was 30,000. Trypsin inhibitor mRNA directed the synthesis of a 23,800 dalton polypeptide as compared to 21,500 daltons for trypsin inhibitor marker protein. Lectin specific polysomes could not be obtained from a soybean variety which lacks detectable lectin protein whereas trypsin inhibitor-specific polysomes were bound by immunoselection. The specificity of the immunoadsorption procedure was confirmed and strongly indicated that the lectinless variety was deficient or substantially reduced in functional lectin mRNA.