Isolation and analysis of a cDNA clone that encodes an alfalfa (Medicago sativa) aspartate aminotransferase |
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Authors: | Michael K Udvardi and Michael L Kahn |
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Institution: | (1) Botanisches Institut der Universit?t, W-5300 Bonn 1, FRG |
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Abstract: | Summary Nitrate reductase (NR) assays revealed a bi-specific NAD(P)H-NR (EC 1.6.6.2.) to be the only nitrate-reducing enzyme in leaves
of hydroponically grown birches. To obtain the primary structure of the NAD(P)H-NR, leaf poly(A)+ mRNA was used to construct a cDNA library in the lambda gt11 phage. Recombinant clones were screened with heterologous gene
probes encoding NADH-NR from tobacco and squash. A 3.0 kb cDNA was isolated which hybridized to a 3.2 kb mRNA whose level
was significantly higher in plants grown on nitrate than in those grown on ammonia. The nucleotide sequence of the cDNA comprises
a reading frame encoding a protein of 898 amino acids which reveals 67%–77% identity with NADH-nitrate reductase sequences
from higher plants. To identify conserved and variable regions of the multicentre electron-transfer protein a graphical evaluation
of identities found in NR sequence alignments was carried out. Thirteen well-conserved sections exceeding a size of 10 amino
acids were found in higher plant nitrate reductases. Sequence comparisons with related redox proteins indicate that about
half of the conserved NR regions are involved in cofactor binding. The most striking difference in the birch NAD(P)H-NR sequence
in comparison to NADH-NR sequences was found at the putative pyridine nucleotide binding site. Southern analysis indicates
that the bi-specific NR is encoded by a single copy gene in birch.
These sequence data appeared in the EMBL/GenBank/DDBJ nucleotide sequence data bases under the accession number X54097 |
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Keywords: | Betula pendula cDNA cloning NAD(P)H-nitrate reductase Sequence comparison |
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