Molecular cloning of a Brassica napus thiohydroximate S-glucosyltransferase gene and its expression in Escherichia coli

A genomic clone encoding a thiohydroximate S-glucosyltransferase (S-GT) was isolated from Brassica napus by library screening with probes generated by PCR using degenerated primers. Its corresponding cDNA was amplified by rap id amplification of cDNA ends (RACE) PCR and also cloned by cDNA library sc reening. The genomic clone was 5 896 bp long and contained a 173-bp intron. At least two copies of the S-GT gene were present in B. napus. The full-le ngth cDNA clone was 1.5 kb long and contained an open reading frame encodin g a 51-kDa polypeptide. The deduced amino acid sequence shared a significan t degree of homology with other glucosyltransferases characterized in other species, including a highly conserved motif within this family of enzymes corresponding to the glucose-binding domain. The recombinant protein was ex pressed in Escherichia coli, and the enzyme activity was tested by a bioche mical assay based on the measure of glucose incorporation. The high thiohyd roximate S-GT activity detected from the recombinant protein confirmed that this clone was indeed a S-glucosyltransferase.