| NADH-nitrate reductase (EC 1.6.6.1) is the predominant form of nitrate reductase in most higher plants including barley (Hordeum vulgare). Here I report the sequencing and analysis of a near full length barley nitrate reductase cDNA and a full length barley genomic nitrate reductase clone. Comparison of the deduced amino acid sequence with nitrate reductase from other plant species shows that the coding region for nitrate reductase is highly conserved. The codon usage for the barley nitrate reductase gene, like most other monocots was found to have a high GC bias in the third codon position. The transcription start site for barley NADH NR was located by primer extension and putative TATA and CAAT boxes have been identified. A sequence located at position 797 upstream from the RNA cap site has been identified by its homology to the E. Coli nitrate induction sequence for the nar operon. The barley genomic clone is 7.2 kb in length, contains a single 1.6 kb intron and extends over 2.5 kb upstream of the translation initiation site.;Analysis of the barley NADH nitrate reductase gene can be facilitated by amplification of specific nitrate reductase fragments from barley genomic DNA using the polymerase chain reaction. A specific region of the nitrate reductase gene from single seedlings of barley was amplified, cloned and sequenced in order to assign apparent allelic differences between the two plant types present in the cv. Steptoe to a specific sequence pattern. Partial sequencing and analysis demonstrated hybrid molecules were produced by the polymerase chain reaction. Production of hybrid molecules in vitro make sequence analysis of polymerase chain reaction products problematic. Recombination may occur in the polymerase chain reaction if two or more similar genes are present as may be the case in polyploids, heterozygotes and multi-gene families or DNA contaminants. |
