Goto S, Akagawa T, Kojima S, Hayakawa T, Yamaya T
Department of Life Science, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan.
Biochim Biophys Acta. 1998 Sep 8;1387(1-2):298-308. doi: 10.1016/s0167-4838(98)00142-3.
Genomic clones for NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14) were obtained from a genomic library of rice (Oryza sativa L. cv. Sasanishki). A genomic clone (lambdaOS42, 14 kb) covered an entire structural gene and a 3.7 kb 5'-upstream region from the first methionine. Another clone (lambdaOS23, 14 kb) contained a 2.8 kb 3'-downstream region from the stop codon. A 7047 bp long clone (lambdaOSR51) consisting of full length cDNA for NADH-GOGAT was isolated from a cDNA library prepared using mRNA from roots of rice seedlings treated with 1 mM NH4Cl for 12 h. The presumed transcribed region (11.7 kb) consisted of 23 exons separated by 22 introns. Rice NADH-GOGAT is synthesized as a 2166 amino acid protein with a molecular mass of 236.7 kDa that includes a 99 amino acid presequence. DNA gel blot analysis suggested that NADH-GOGAT occurred as a single gene in rice. Primer extension experiments map the transcription start of NADH-GOGAT to identical positions. The 3. 7 kb 5'-upstream region was able to transiently express a reporter gene in cultured rice cells. Putative motifs related to the regulation of NADH-GOGAT gene expression were looked for within the 5'-upstream region by database.
从水稻（Oryza sativa L. cv. Sasanishki）的基因组文库中获得了依赖NADH的谷氨酸合酶（NADH-GOGAT；EC 1.4.1.14）的基因组克隆。一个基因组克隆（λOS42，14 kb）覆盖了整个结构基因以及从第一个甲硫氨酸起的3.7 kb 5'上游区域。另一个克隆（λOS23，14 kb）包含从终止密码子起的2.8 kb 3'下游区域。从用1 mM NH4Cl处理12小时的水稻幼苗根中提取的mRNA构建的cDNA文库中分离出一个由NADH-GOGAT全长cDNA组成的7047 bp长的克隆（λOSR51）。推测的转录区域（11.7 kb）由23个外显子组成，被22个内含子隔开。水稻NADH-GOGAT作为一种分子量为236.7 kDa的2166个氨基酸的蛋白质合成，其中包括一个99个氨基酸的前导序列。DNA凝胶印迹分析表明，NADH-GOGAT在水稻中以单基因形式存在。引物延伸实验将NADH-GOGAT的转录起始位点定位到相同位置。3.7 kb的5'上游区域能够在培养的水稻细胞中瞬时表达报告基因。通过数据库在5'上游区域寻找与NADH-GOGAT基因表达调控相关的假定基序。
