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Molecular cloning of a Brassica napus thiohydroximate S ‐glucosyltransferase gene and its expression in Escherichia coli
Author(s) -
Marillia ElizabethFrance,
MacPherson Jim M.,
Tsang Edward W. T.,
Van Audenhove Katrien,
Keller Wilf A.,
GrootWassink Jan W. D.
Publication year - 2001
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1034/j.1399-3054.2001.1130204.x
Subject(s) - complementary dna , biology , microbiology and biotechnology , open reading frame , cdna library , gene , genomic library , glucosyltransferase , molecular cloning , escherichia coli , recombinant dna , cloning (programming) , homology (biology) , genomic dna , genetics , clone (java method) , rapid amplification of cdna ends , peptide sequence , computer science , programming language
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.