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 oligonucleotides 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 predicted relative molecular mass of 65810 Da, an isoelectric point of 6.4, and a net charge of -7.2 at pH 7.0. The amino acid sequence of the protein encoded by PVAS2 is very similar to that encoded by the soybean SAS2 asparagine synthetase gene. The amino-terminal residues of the predicted PVAS2 protein 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 blot revealed that the PVAS2 transcript accumulates to a high level in roots and, 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.
Osuna
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Departamento de Bioquímica y Biología Molecular, e Instituto Andaluz de Biotecnología, Facultad de Ciencias, Universidad de Córdoba, Avda. San Alberto Magno, s/n. 14071, Córdoba, Spain.
Gálvez
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Pineda
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Aguilar
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D000595:Amino Acid Sequence; D001217:Aspartate-Ammonia Ligase / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000302:isolation & purification; D007887:Fabaceae / Q000235:genetics*; D008969:Molecular Sequence Data; D010946:Plants, Medicinal; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment
D018076:DNA, Complementary; D012333:RNA, Messenger; D001217:Aspartate-Ammonia Ligase