Identification and expression of a cDNA clone encoding aspartate aminotransferase in carrot

• Published in: Plant physiology (Bethesda) 1992-09, Vol.100 (1), p.374-381
• Main Authors: TURANO, F. J, WEISEMANN, J. M, MATTHEWS, B. F
• Format: Article
• Language: English
• Subjects: Agronomy. Soil science and plant productions; Amino acids; arn mensajero; arn messager; aspartate aminotransferase; aspartato aminotransferasa; Biological and medical sciences; cell culture; Cell culture techniques; citoplasma; Complementary DNA; cultivo de celulas; culture de cellule; Cultured cells; cytoplasm; cytoplasme; daucus carota; DNA; Enzymes; expresion genica; expression des genes; Fundamental and applied biological sciences. Psychology; Gels; gene; gene expression; genes; messenger rna; Metabolism; Molecular Biology and Gene Regulation; nucleotide sequence; Nucleotides; Plant physiology and development; Polymerase chain reaction; racine; raices; RNA; roots; secuencia nucleica; sequence nucleique
• ISSN: 0032-0889; 1532-2548
• DOI: 10.1104/pp.100.1.374

A full-length cDNA clone encoding aspartate aminotransferase (AAT) has been identified from a carrot root cDNA library. Degenerate oligo primers were synthesized from the known amino acid sequence of AAT form I from carrot (Daucus carota L. cv Danvers). These primers were utilized in a polymerase chain reaction to amplify a portion of a carrot AAT gene from first strand cDNA synthesized from poly(A)+ RNA isolated from 5-d-old cell suspension cultures. The resulting 750-bp fragment was cloned, mapped, and sequenced. The cloned fragment, mpAAT1, was used as a probe to identify a full-length cDNA clone in a library constructed from poly(A)+ RNA isolated from carrot roots. A 1.52-kb full-length clone, AAT7, was isolated and sequenced. AAT7 has 54% nucleotide identity with both the mouse cytoplasmic and mitochondrial AAT genes. The deduced amino acid sequence has 52 and 53% identity with the deduced amino acid sequences of mouse cytoplasmic and mitochondrial AAT genes, respectively. Further analysis of the sequence data suggests that AAT7 encodes a cytoplasmic form of carrot AAT; the evidence includes the (a) absence of a transit or signal sequence, (b) lack of "m-residues," or invariant mitochondrial residues, in the carrot AAT sequence, and (c) high degree of sequence similarity with the amino acid sequence previously obtained for form I of carrot, a cytoplasmic isoenzyme. High- and low-stringency hybridizations to Southern blots of carrot nuclear DNA with AAT7 show that AAT is part of a small multigene family. Northern blot analysis of AAT7 suggests that AAT is expressed throughout cell culture up to 7 d and is highly expressed in roots but not in leaves.