Vaucheret H, Vincentz M, Kronenberger J, Caboche M, Rouzé P
Laboratoire de Biologie Cellulaire, INRA-Centre de Versailles, France.
Mol Gen Genet. 1989 Mar;216(1):10-5. doi: 10.1007/BF00332224.
The two structural genes encoding tobacco nitrate reductases (NR) were isolated from tobacco genomic libraries constructed in lambda EMBL phages. Two independent genomic clones of 12.6 and 13.5 kbp, respectively, cross-hybridizing with a partial tobacco NR cDNA probe, were further characterized. Southern blot experiments were performed with the NR cDNA probe on genomic DNA derived from Nicotiana tabacum and from the ancestors of tobacco, N. sylvestris and N. tomentosiformis. They showed that the larger clone, referred to as nia-1, was related to the N. tomentosiformis parent, and the smaller one, referred to as nia-2, to the N. sylvestris parent. Both homeologous genes were found to be expressed in tobacco. The sequence of the gene nia-2, from which the cDNA previously cloned is derived, was determined. It encodes a 904 amino acid protein. Three intervening sequences were found interspersed with the coding sequence of the enzyme. The precise location of the transcription initiation site on the structural gene was mapped by primer extension experiments. A TATA consensus sequence was detected 32 bp upstream from the transcription initiation site. The leader sequence of the transcript is 138 nucleotides long and a stable secondary structure involving the translation initiation site has been proposed. The amino acid sequence of tobacco NR deduced from the nucleotide sequence of the gene shows that heme and FAD binding domains occupy the entire C-terminal moiety of the polypeptide. The remaining N-terminal part of the protein should thus carry the catalytic site of nitrate reduction by the molybdenum cofactor.
从构建于λEMBL噬菌体中的烟草基因组文库中分离出了编码烟草硝酸还原酶（NR）的两个结构基因。分别对两个独立的12.6kbp和13.5kbp的基因组克隆进行了进一步表征，这两个克隆与部分烟草NR cDNA探针发生交叉杂交。用NR cDNA探针对来自烟草（Nicotiana tabacum）及其祖先野生烟草（N. sylvestris）和绒毛状烟草（N. tomentosiformis）的基因组DNA进行了Southern杂交实验。结果表明，较大的克隆（称为nia - 1）与绒毛状烟草亲本相关，较小的克隆（称为nia - 2）与野生烟草亲本相关。发现这两个同源基因在烟草中均有表达。确定了先前克隆的cDNA所源自的nia - 2基因的序列。它编码一种含904个氨基酸的蛋白质。发现三个间隔序列穿插于该酶的编码序列中。通过引物延伸实验确定了结构基因上转录起始位点的精确位置。在转录起始位点上游32bp处检测到一个TATA共有序列。转录本的前导序列长138个核苷酸，并提出了一种涉及翻译起始位点的稳定二级结构。从该基因的核苷酸序列推导得到的烟草NR氨基酸序列表明，血红素和FAD结合结构域占据了多肽的整个C末端部分。因此，该蛋白质其余的N末端部分应携带钼辅因子进行硝酸盐还原的催化位点。
