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

Halim bin Hamat, . Molecular cloning and characterization of nitrate reductase genes in rice (Oryza sativa L.). pp. 1-11. ISSN 0128-0686

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 EcoRI 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 EcoRI 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 ARI and lambda BRI respectively. The 8.2 kbp EcoRI/BamHI fragment of lambda ARI and the 13.5 kbp EcoRI/BamHI fragment of lambda BRI were homologous to the barley cDNA probe and were subcloned 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. The 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.

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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 EcoRI 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 EcoRI 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 ARI and lambda BRI respectively. The 8.2 kbp EcoRI/BamHI fragment of lambda ARI and the 13.5 kbp EcoRI/BamHI fragment of lambda BRI were homologous to the barley cDNA probe and were subcloned 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. The 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.

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Item Type:	Article
Additional Information:	11 ill.; 31 ref. Summary (En Malay)
AGROVOC Term:	ORYZA SATIVA
AGROVOC Term:	NITRATOS
AGROVOC Term:	OXIDORREDUCTASAS
AGROVOC Term:	GENES/ CLONACION
Depositing User:	Ms. Norfaezah Khomsan
Last Modified:	24 Apr 2025 05:56
URI:	http://webagris.upm.edu.my/id/eprint/20315

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