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 characteristics of an alpha-glucosidase. Phylogenetic analysis of the deduced polypeptide encoded by this cDNA demonstrated that the most similar sequences were alpha-glucosidases and alpha-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-alpha-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-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 severely down-regulated. Despite this, no decrease in total extractable alpha-glucosidase and alpha-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.
Taylor
M A
MA
Unit of Plant Biochemistry, Scottish Crop Research Institute, Invergowrie, Dundee, UK. mtaylo@scri.sari.ac.uk
Ross
H A
HA
McRae
D
D
Wright
F
F
Viola
R
R
Davies
H V
HV
D003001:Cloning, Molecular; D018076:DNA, Complementary; D015536:Down-Regulation; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010802:Phylogeny; D030821:Plants, Genetically Modified; D029702:Schizosaccharomyces pombe Proteins; D011198:Solanum tuberosum / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D014995:Xylosidases / Q000145:classification / Q000235:genetics / Q000378:metabolism; D000520:alpha-Glucosidases / Q000145:classification / Q000235:genetics* / Q000378:metabolism
D018076:DNA, Complementary; C102110:Mal2 protein, S pombe; D009687:Nuclear Proteins; D029702:Schizosaccharomyces pombe Proteins; D014995:Xylosidases; D000520:alpha-Glucosidases