Copy-DNA cloning and characterisation of a potato α-glucosidase: expression in Esherichia coli and effects of down-regulation in transgenic potato.

Taylor, Mark A.; Ross, Heather A.; McRae, Diane; Wright, Frank; Viola, Roberto; Davies, Howard V.

Polymerase chain reaction-based methodology was used to obtain a cDNA clone (MAL2) from potato (Solanum tuberosum L.) with the sequence characteristics of an α-glucosidase. Phylogenetic analysis of the deduced polypeptide encoded by this cDNA demonstrated that the most similar sequences were α-glucosidases and α-xylosidases of plant origin. The MAL2 cDNA was expressed in Escherichia coli and the recombinant MAL2 protein was affinity-purified. MAL2 catalysed the hydrolysis of a range of maltooligomers and p-nitrophenyl-α-D-glucopyranoside with a pH optimum of 5.5–5.7. The substrate with the lowest Km value was maltotetraose (3.7 mM). The MAL2 expression product did not catalyse the hydrolysis of xyloglucan oligosaccharides, p-nitrophenyl-α-D-xylopyranoside or gelatinised potato starch. MAL2 was down-regulated in transgenic potato plants using an antisense approach. In several independent transgenic antisense lines, MAL2 expression was severely down-regulated. Despite this, no decrease in total extractable α-glucosidase and α-xylosidase activity could be detected in tissues from the transgenic plants. In glasshouse trials, no visible phenotype, change in tuber yield or carbohydrate content was associated with MAL2 down-regulation. The implications of these results are discussed.

POLYMERASE chain reaction; DNA polymerases; POTATOES; ESCHERICHIA coli; HYDROGEN-ion concentration; ORGANIC compounds

Planta: An International Journal of Plant Biology, 2001, Vol 213, Issue 2, p258

0032-0935

Academic Journal

10.1007/s004250000502