Kalamaki Mary S, Alexandrou Dimitris, Lazari Diamanto, Merkouropoulos Georgios, Fotopoulos Vasileios, Pateraki Irene, Aggelis Alexandros, Carrillo-López Armando, Rubio-Cabetas Maria J, Kanellis Angelos K
Division of Pharmacognosy-Pharmacology, Department of Pharmaceutical Sciences, Aristotle University of Thessaloniki, Thessaloniki, Greece.
J Exp Bot. 2009;60(6):1859-71. doi: 10.1093/jxb/erp072. Epub 2009 Apr 8.
A single copy of the N-acetyl-L-glutamate synthase gene (SlNAGS1) has been isolated from tomato. The deduced amino acid sequence consists of 604 amino acids and shows a high level of similarity to the predicted Arabidopsis NAGS1 and NAGS2 proteins. Furthermore, the N-terminus ArgB domain and the C-terminus ArgA domain found in SlNAGS1 are similar to the structural arrangements that have been reported for other predicted NAGS proteins. SlNAGS1 was expressed at high levels in all aerial organs, and at basic levels in seeds, whereas it was not detected at all in roots. SlNAGS1 transcript accumulation was noticed transiently in tomato fruit at the red-fruit stage. In addition, an increase of SlNAGS1 transcripts was detected in mature green tomato fruit within the first hour of exposure to low oxygen concentrations. Transgenic Arabidopsis plants have been generated expressing the SlNAGS1 gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Three homozygous transgenic lines expressing the transgene (lines 1-7, 3-8, and 6-5) were evaluated further. All three transgenic lines showed a significant accumulation of ornithine in the leaves with line 3-8 exhibiting the highest concentration. The same lines demonstrated higher germination ability compared to wild-type (WT) plants when subjected to 250 mM NaCl. Similarly, mature plants of all three transgenic lines displayed a higher tolerance to salt and drought stress compared to WT plants. Under most experimental conditions, transgenic line 3-8 performed best, while the responses obtained from lines 1-7 and 6-5 depended on the applied stimulus. To our knowledge, this is the first plant NAGS gene to be isolated, characterized, and genetically modified.
已从番茄中分离出N-乙酰-L-谷氨酸合酶基因(SlNAGS1)的单拷贝。推导的氨基酸序列由604个氨基酸组成,与预测的拟南芥NAGS1和NAGS2蛋白具有高度相似性。此外,在SlNAGS1中发现的N端ArgB结构域和C端ArgA结构域与其他预测的NAGS蛋白所报道的结构排列相似。SlNAGS1在所有地上器官中高水平表达,在种子中表达水平较低,而在根中未检测到。在红果期番茄果实中短暂观察到SlNAGS1转录本积累。此外,在暴露于低氧浓度的第一小时内,成熟绿番茄果实中检测到SlNAGS1转录本增加。已构建在花椰菜花叶病毒(CaMV)35S启动子控制下表达SlNAGS1基因的转基因拟南芥植株。进一步评估了三个表达转基因的纯合转基因系(系1-7、3-8和6-5)。所有三个转基因系的叶片中鸟氨酸均有显著积累,其中系3-8的浓度最高。与野生型(WT)植株相比,这三个系在250 mM NaCl处理下表现出更高的发芽能力。同样,与WT植株相比,所有三个转基因系的成熟植株对盐和干旱胁迫表现出更高的耐受性。在大多数实验条件下,转基因系3-8表现最佳,而系1-7和6-5的反应则取决于所施加的刺激。据我们所知,这是第一个被分离、表征和基因改造的植物NAGS基因。