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 rapid amplification of cDNA ends (RACE) PCR and also cloned by cDNA library screening. 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-length cDNA clone was 1.5 kb long and contained an open reading frame encoding a 51-kDa polypeptide. The deduced amino acid sequence shared a significant 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 expressed in Escherichia coli, and the enzyme activity was tested by a biochemical assay based on the measure of glucose incorporation. The high thiohydroximate S-GT activity detected from the recombinant protein confirmed that this clone was indeed a S-glucosyltransferase.
Marillia
Elizabeth-France
EF
Plant Biotechnology Institute, National Research Council of Canada, 110 Gymnasium Place, Saskatoon, SK S7N 0W9, Canada Performance Plants Inc., 410 Downey Road, Saskatoon, SK S7N 4R2, Canada Aventis CropScience N.V., Jozef Plateaustraat 22, B-9000 Gent, Belgium GNC Bioferm Inc., 1220 Wilder Road, Cobble Hill, BC V0R 1L0, Canada.
MacPherson
Jim M.
JM
Tsang
Edward W. T.
EW
Van Audenhove
Katrien
K
Keller
Wilf A.
WA
GrootWassink
Jan W. D.
JW