Cloning and Sequence Analysis of the Nucleotide-Binding Domain of an Alpha-Glucan, Water Dikinase Gene from Cassava (Manihot esculenta Crantz)

Abstract

Several key genes involved in starch biosynthesis have been identified in cassava. However, while phosphorylation has been recognized as an essential step in starch metabolism in higher plants, the underlying gene(s) in cassava have not been isolated so far. To gain insights into starch phosphorylation in cassava, we produced three genomic clones encoding a fragment of an α-glucan, water dikinase (GWD), the primary enzyme required for starch phosphorylation. Sequence analysis showed that each of the clones contained the nucleotide-binding domain of the C-terminus of plant GWDs. The three genomic gwd clones had 98.8% homology at both nucleotide and amino acid levels and represented two distinct allelic sequences with two amino acid substitutions. The shorter clone, pOYE308-1, was 561 base pairs long and encoded a polypeptide of 106 amino acids with a predicted molecular weight of 180.3kDa. Two putative introns were identified in each of the gwd clones. Phylogenetic analysis of the cassava GWD sequences with other plant GWDs revealed that the cassava GWD belongs to the same group as that of castor bean, potato, tomato and tobacco. These resources add to the current knowledge base of starch metabolism in cassava and expand the molecular tool box for starch modification in this important tropical root crop.