370 Isolation of a partial isoamylase gene from a wheat cDNA library.

S. NETRPHAN (1,2), R. L. Khandelwal (2), and R. N. Chibbar (1). (1) Plant Biotechnology Institute, National Research Council Canada, 110 Gymnasium Place, Saskatoon, SK, Canada S7N 0W9; (2) Department of Biochemistry, College of Medicine, University of Saskatchewan, 107 Wiggins Road, Saskatoon, SK, Canada S7N 5E5.

Starch debranching enzyme activity has been ascribed either to isoamylase (EC 3.2.1.68) or pullulanase (EC 3.2.1.41) based on the differences in their substrate specificities. Besides the well-known function of starch debranching enzymes in starch degradation, these enzymes have recently been implicated in the synthesis of amylopectin, the highly branched glucan polymer in starch. Starch debranching enzymes are members of the amylolytic enzyme family, which have four conserved domains predicted for possible active sites. Two oligonucleotide primers were synthesized to contain sequences derived from the first (5’- GGGATGTTGTCTTCAATCATAC-3’) and fourth (5’-GTCCATCGTGTGCACATACAA-3’) conserved regions of maize isoamylase cDNA. These two oligonucleotide primers in a polymerase chain reaction amplified a 619 bp fragment from a wheat (Triticum aestivum cv. Fielder) cDNA library constructed with RNA from a developing kernel. The amplified DNA fragment showed 88% similarity at the nucleotide level and 93% similarity at the amino acid level to maize isoamylase cDNA. Screening of 1 × 10(^6) plaque forming units (pfu) with the radio-labeled isolated DNA fragment yielded fifteen positive clones. The longest cDNA was 2.2 kb and exhibited higher sequence similarity to the isoamylase cDNA from rice than to that from maize. Results on the characterization of the wheat isoamylase gene and its expression during grain development will be presented.