Analysis of barley nitrate reductase cDNA and genomic clones

Abstract

Barley nitrate reductase cDNA and genomic clones were isolated by homology with the barley nitrate reductase cDNA clone bNRp10 and sequenced. This is the first reported analysis of a full-length nitrate reductase gene and its corresponding cDNA in the same species. The longest cDNA clone extends to within 9 bp of the ATG start codon and the sequence is similar to that reported for the higher plant NR sequences. As expected, the amino acid sequence of barley nitrate reductase is more related closely to the rice (84% homology) than to the Arabidopsis (62%) sequence. Four different polyA addition sites were identified from sequence analysis of nine barley NR cDNA clones. A 7.3 kb region of a genomic recombinant lambda clone was subcloned as two contiguous BamHI fragments into p Bluescript, designated pMJ7 and pMJ8, and sequenced. These clones include the entire nitrate reductase coding region, one large intron, 2.7 kb of untranslated sequence 5' to the translation start codon and 0.25 kb 3' to the translation termination codon. The mRNA cap site was identified as a cytosine, 111 bases upstream of the ATG translation start codon. The putative CAAT and TATA boxes were identified at -115 and -33 bp, respectively, with the mRNA cap site designated as +1. The barley nitrate reductase gene coding region strongly favors G or C in the third codon position.