Molecular cloning and characterization of nitrate reductase genes in rice (Oryza sativa L.)

H. B. Hamat

Abstract

A barley Nitrate Reductase cDNA clone was used as a hybridization probe to investigate the nitrate reductase gene organization in rice cv. M 201 (subspecies japonica) and Labelle (subspecies indica). Three EcoRl bands (18, 14, and 12 kbp) were detected in cv. Labelle DNA and four (18, 14, 10 and 3 kbp) in cv. M 201. Southern dot blot analysis indicated that there were eight nitrate reductase gene copies in cv. Labelle and six in cv. M 201 per haploid genome. A partial EcoRl digested cv. M 201 DNA library in lambda Charon 35 vector was screened in order to isolate and characterize the rice nitrate reductase genes. Clones containing the 14 kbp and 18 kbp EcoRI fragments were isolated and designated lambda AR1 and lambda BR1 respectively. The 8.2 kbp EcoRI/BamHI fragment of lambda AR1 and the 13.5 kbp EcoRI/BamHl fragment of lambda BR1 were homologous to the barley cDNA probe, and were subeloned into pUC8 to form recombinant plasmids pHBH1 and pHBH2, respectively. The restriction endonuclease maps of pHBH1 and pHBH2 clones were constructed, and the 3' end of the rice nitrate reductase gene in each recombinant plasmid was determined. The two clones were different. pHBH1 contained unique DNA sequence of 5.6 kbp which was flanked by highly repetitive DNA sequence at the 3' but not at the 5' end. This clone might not contain the 5' end of the gene. However, pHBH2 contained unique sequences of 6.5 kbp flanked by highly repetitive DNA sequences at both the 3' and 5' ends. These results suggested that pHBH2 clone contains a complete nitrate reductase gene. Results from hybridization of each DNA fragment from pHBH1 and pHBH2 to total
RNA isolated from nitrate induced rice seedling further supported the conclusion that pHBH2 clone contained a complete nitrate reductase gene while pHBH1 did not.

Full Text (610 KB)