ISOLATION AND CHARACTERIZATION OF A MONOFUNCTIONAL ASPARTOKINASE cDNA FROM SOYBEAN

Soybean and Alfalfa Research Laboratory, US Department of Agriculture, Agricultural Research Service, Beltsville MD, 20705 USA

We have amplified a PCR fragment from a cDNA library prepared from PolyA RNA isolated from Glycine max cv. Century grown for 6 days in the dark. This fragment has homology to two Arabidopsis thaliana monofunctional aspartokinase clones. The amino acid translation of this soybean clone has approximately 80% identity to the amino acid sequence of either of the Arabidopsis clones. Reverse transcription of G max cv. Century PolyA RNA yielded a PCR fragment coding for an 88 amino acid transit peptide. Comparison of the amino acid sequence of this aspartokinase to other monofunctional and bifunctional aspartokinases showed a higher degree of homology to the E coli monofunctional isoform than to the bifunctional isoform from soybean suggesting that the monofunctional and bifunctional isoforms constitute two distinct lines separating early in evolution. This soybean monofunctional clone contains the same three highly conserved amino acid motifs present in all aspartokinases sequenced so far. However, there are slight differences in these motifs between the monofunctional and bifunctional isoforms. A northern blot of PolyA RNA isolated from soybean cotyledons and leaves grown in the dark and light for 4 or 8 days was hybridized with the soybean aspartokinase clone. In all cases a single band of 2.25 kb was obtained. The level of transcript appeared to be higher in tissues of light grown seedlings in all cases.