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Molecular cloning and characterization of a cDNA encoding soybean ferric leghemoglobin reductase
Abstract
A cDNA encoding soybean (Glycine max (L.) Merr) ferric leghemoglobin reductase (FLbR) has been cloned and characterized. A group of highly degenerate oligonucleotides deduced from the N-terminal amino acid sequence of FLbR was used to prime the polymerase chain reaction (PCR) on soybean nodule mRNA and cDNA. A full clone of FLbR cDNA was isolated by screening a $\lambda$gt11 soybean nodule cDNA library using the specific PCR-amplified FLbR cDNA fragment as a probe. The cDNA contained about 1.8 kilobases and had a coding sequence of 523 amino acids that included a putative 30-residue signal peptide and a 494-residue mature protein with a predicted molecular mass of 55,729 daltons. Computer-aided analysis of the deduced FLbR amino acid sequence showed considerable homology (varied from 20% to 50% with enzymes and species) to dihydrolipoamide dehydrogenase (EC 1.8.1.4), glutathione reductase (EC 1.6.4.2), mercuric reductase (EC 1.16.1.1), and trypanothione reductase (EC 1.6.4.8) in a superfamily of pyridine nucleotide-disulfide oxidoreductases from various organisms. Northern-blot analysis of RNA using FLbR cDNA as a probe showed that the FLbR gene was expressed in soybean nodules, leaves, roots, and stems, with a greater level of expression in nodules and leaves than in roots and stems. Southern-blot analysis of the genomic DNA showed the presence of two homologous FLbR genes in the soybean genome. The cloned soybean FLbR cDNA has been subcloned into an expression plasmid pTrcHis C and overexpressed in Escherichia coli. The overexpressed FLbR was purified by two steps of column chromatography: ammonium sulfate gradient fractionation by Sepharose 6B and affinity separation by Proboud resin, and characterized by electrophoretic analysis and immunoblotting analysis with antisera against FLbR from soybean root nodules. The recombinant FLbR protein was proved to be catalytically active and fully functional. UV-visible absorption and fluorescence spectra, fluorescence polarization and anisotropy, cofactor (NADH and FAD) binding features, and the catalytic, kinetic and dynamic properties was compared between the cloned FLbR and the native FLbR purified from soybean root nodules and the pig heart and yeast dihydrolipoamide dehydrogenases.
Subject Area
Biochemistry|Molecular biology
Recommended Citation
Ji, Lin, "Molecular cloning and characterization of a cDNA encoding soybean ferric leghemoglobin reductase" (1993). ETD collection for University of Nebraska-Lincoln. AAI9402393.
https://digitalcommons.unl.edu/dissertations/AAI9402393