Cloning of a nitrate reductase inactivator (NRI) cDNA from Spinacia oleracea L. and expression of mRNA and protein of NRI in cultured spinach cells.
The spinach ( Spinacia oleracea L. (cv. Hoyo) nitrate reductase inactivator (NRI) is a novel protein that irreversibly inactivates NR. Using degenerate primers based on an N-terminal amino acid sequence of NRI purified from spinach leaves and a cDNA library, we isolated a full-length NRI cDNA from spinach that contains an open reading frame encoding 479 amino acid residues. This protein shares 67.4% and 51.1-68.3% amino acid sequence similarities with a nucleotide pyrophosphatase (EC 3.6.1.9) from rice and three types of the nucleotide pyrophosphatase-like protein from Arabidopsis thaliana, respectively. Immunoblot analysis revealed that NRI was constitutively expressed in suspension-cultured spinach cells; however, its expression level is quite low in 1-day-subcultured cells. Moreover, northern blot analysis indicated that this expression was regulated at the mRNA level. These results suggest that NRI functions in mature cells.
Sonoda
Masatoshi
M
Department of Bioproduction Science, Faculty of Horticulture, Chiba University, 271, Matsudo, Chiba, Japan.
Ide
Hiroaki
H
Nakayama
Shinya
S
Sasaki
Asako
A
Kitazaki
Shinei
S
Sato
Takahide
T
Nakagawa
Hiroki
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003429:Cross Reactions / Q000276:immunology; D018076:DNA, Complementary / Q000235:genetics*; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant / Q000235:genetics*; D008969:Molecular Sequence Data; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000037:antagonists & inhibitors*; D010940:Plant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D018724:Spinacia oleracea / Q000166:cytology / Q000235:genetics* / Q000378:metabolism
D018076:DNA, Complementary; D010940:Plant Proteins; D012333:RNA, Messenger; D009565:Nitrate Reductases; D050901:Nitrate Reductase