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It was previously reported that the rice nitrate reductase (NR)-deficient mutant M819 (wild type: cv. Norin 8) Iacks the functional heme domain of NADH-NR polypeptide possibly due to a point mutation or small deletion. In this paper we were able to locate the mutation in the NR apoenzyme gene. Northern blot analysis using a rice NADH-NR genomic clone as a probe showed that the size of NR mRNA of M819 was the same as that of the wild type, suggesting that the mutation did not involve a large deletion or insertion in the NADH-NR coding sequence. The DNA fragment containing the coding region of NADH-NR heme domain was amplified by PCR from M 819 and Norin 8. The alignment of nucleotide sequences of the domain revealed that 3 nucleotides (GTC) encoding one valine residue (Val-561 or Val-562) were deleted in M819. Thus, it was demonstrated that there was a point mutation in the coding region of NADH-NR heme dornain in the mutant M819., イネには2つのタイプの硝酸還元酵素(NR),即ちNADH-NR及びNAD(P)H-NRがある。イネ突然変異体M819(野生型は農林8号)ではNADH-NRの活性が欠失しており,さらにNR部分活性の測定結果からM819のNADH-NR遺伝子のヘムドメインコーディング領域には点突然変異あるいは微小な欠失が生じていると予想されている(Hasegawa et al.1992)。本実験ではその検証を試みた。イネNADH-NR遺伝子pHBH1クローンをプローブに用いてノーザンブロット解析を行ったところM819及び農林8号の両者に約3kbのパンドが検出された(図1)。約3kb以外のパンドがM819で検出されなかったことから,M819のNR遺伝子のエクソン内にはノーザンブロットで確認できるような大きな構造変異は生じていないと考えられた。次にイネ品種M201からクローニングされたNADH-NR遺伝子の塩基配列をもとにプライマーを設計し,M819及び農林8号のDNAからNADH-NRヘムドメインを含む約700bpのDNA断片をそれぞれPCRにより増幅した(図2)。得られたPCR産物はpBluescriptIISK+にクローニングした。農林8号由来のクローンを使用してPCR産物とpHBH1クローンとの相同性をサザンブロット及びノーザンブロット法により確認した後(図3),M819及び農林8号のNADH-NRヘムドメインの塩基配列を決定した(図4)。農林8号の塩基配列は既知の配列と同一であったが,19では2つ並んだバリン残基(Val-561及びVal-562)のうちどちらか一方に相当する3塩基対の欠失が認められた。またこの点突然変異は,PCR法を用いる事により容易に検出することができた(図5)。

Transgenic plants of Arabidopsis bearing the spinach (Spinacia oleracea) nitrite reductase (NiR, EC 1.7.7.1) gene that catalyzes the six-electron reduction of nitrite to ammonium in the second step of the nitrate assimilation pathway were produced by use of the cauliflower mosaic virus 35S promoter and nopaline synthase terminator. Integration of the gene was confirmed by a genomic polymerase chain reaction (PCR) and Southern-blot analysis; its expression by a reverse transcriptase-PCR and two-dimensional polyacrylamide gel electrophoresis western-blot analysis; total (spinach + Arabidopsis) NiR mRNA content by a competitive reverse transcriptase-PCR; localization of NiR activity (NiRA) in the chloroplast by fractionation analysis; and NO(2) assimilation by analysis of the reduced nitrogen derived from NO(2) (NO(2)-RN). Twelve independent transgenic plant lines were characterized in depth. Three positive correlations were found for NiR gene expression; between the total NiR mRNA and total NiR protein contents (r = 0.74), between the total NiR protein and NiRA (r = 0.71), and between NiRA and NO(2)-RN (r = 0.65). Of these twelve lines, four had significantly higher NiRA than the wild-type control (P0.01), and three had significantly higher NO(2)-RN (P0.01). Each of the latter three had one to two copies of spinach NiR cDNA per haploid genome. The NiR flux control coefficient for NO(2) assimilation was estimated to be about 0.4. A similar value was obtained for an NiR antisense tobacco (Nicotiana tabacum cv Xanthi XHFD8). The flux control coefficients of nitrate reductase and glutamine synthetase were much smaller than this value. Together, these findings indicate that NiR is a controlling enzyme in NO(2) assimilation by plants.