RT-PCR cloning, characterization and mRNA expression analysis of a cDNA encoding a type II asparagine synthetase in common bean

Following a RT-PCR strategy based on the design of degenerate oligonucleoti des resembling conserved domains of asparagine synthetase (AS; EC 6.3.5.4), we isolated a 2 kb cDNA clone (PVAS2) from root tissue of the common bean (Phaseolus vulgaris). PVAS2 encodes a protein of 584 amino acids with a pre dicted relative molecular mass of 65 810 Da, an isoelectric point of 6.4, a nd a net charge of -7.2 at pH 7.0. The amino acid sequence of the protein e ncoded by PVAS2 is very similar to that encoded by the soybean SAS2 asparag ine synthetase gene. The amino-terminal residues of the predicted PVAS2 pro tein are identical to the amino acids that constitute the glutamine-binding (GAT) domain of AS from other plant species, which suggests that the PVAS2 cDNA encodes a type II glutamine-dependent form of asparagine synthetase. Southern blot analysis indicates that the common bean AS is part of a small family composed of at least two genes. Expression analysis by Northern blo t revealed that the PVAS2 transcript accumulates to a high level in roots a nd, to a lesser extent, in nodules and developing pods. Accumulation of the PVAS2 transcript in the root seems to be negatively regulated by light and sucrose, and positively regulated by nitrate. (C) 1999 Elsevier Science B. V. All rights reserved.