MOLECULAR-CLONING AND EXPRESSION OF 2 CDNAS ENCODING ASPARAGINE SYNTHETASE IN SOYBEAN

Two cDNA clones (SAS1 and SAS2) encoding different isoforms of asparag ine synthetase (AS; EC 6.3.5.4) were isolated. Their DNA sequences wer e determined and compared. The amino-terminal residues of the predicte d SAS1 and SAS2 proteins were identical to those of the glutamine bind ing domain of AS from pea, asparagus, Ambidopsis and human, suggesting that SAS1 and SAS2 cDNAs encode the glutamine-dependent form of AS. T he open reading frames of SAS1 and SAS2 encode a protein of 579 and 58 1 amino acids with predicted molecular weights of 65182 and 65608 Da r espectively. Similarity of the deduced amino acid sequences of SAS1 an d SAS2 with other known AS sequences were 92% and 93% for pea AS1; 91% and 96% for pea AS2; 88% and 91% for asparagus; 88% and 90.5% for Amb idopsis; 70.5% and 72.5% for E. coli asnB and 61% and 63% for man. A p lasmid, pSAS2E, was constructed to express the soybean AS protein in E scherichia coli. Complementation experiments revealed that the soybean AS protein was functional in E. coli. Southern blot analysis indicate d that the soybean AS is part of a small gene family. AS transcript wa s expressed in all tissues examined, but higher levels were seen in st em and root of light-grown tissue and leaves of dark-treated tissue.