Copy-DNA cloning and characterisation of a potato alpha-glucosidase: expression in Escherichia coli and effects of down-regulation in transgenic potato

Polymerase chain reaction-based methodology was used to obtain a cDNA clone (MAL2) from potato (Solanum tuberosum L.) with the sequence characteristic s of an alpha -glucosidase. Phylogenetic analysis of the deduced polypeptid e encoded by this cDNA demonstrated that the most similar sequences were a- glucosidases and alpha -xylosidases of plant origin. The MAL2 cDNA was expr essed in Escherichia coli and the recombinant MAL2 protein was affinity-pur ified. MAL2 catalysed the hydrolysis of a range of maltooligomers and p-nit rophenyl-alpha -D-glucopyranoside with a pH optimum of 5.5-5.7. The substra te with the lowest K-m value was maltotetraose (3.7 mM). The MAL2 expressio n product did not catalyse the hydrolysis of xyloglucan oligosaccharides, p -nitrophenyl-alpha -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 severe ly down-regulated. Despite this, no decrease in total extractable alpha -gl ucosidase and alpha -xylosidase activity could be detected in tissues from the transgenic plants. In glasshouse trials, no visible phenotype, change i n tuber yield or carbohydrate content was associated with MAL2 down-regulat ion. The implications of these results are discussed.