Wang Li-Qun, Wang Yong, Dong Ying, Wang Wen-Bing
Institute of Life Sciences, School of Biological & Environmental of Engineering, Jiangsu University, Zhenjiang 212013, China.
Sheng Wu Gong Cheng Xue Bao. 2003 Sep;19(5):632-5.
Excessive nitrate accumulated in plants affects vegetable quality severely and excessive nitrate ingestion would do harm to human health. Assimilatory NADH: nitrate reductase (NR, EC 1.6.6.1), a complex Mo-pterin-, cytochrome b(557)- and FAD-containing protein, catalyzes the regulated and rate-limiting step in the utilization of inorganic nitrogen by higher plants. Enhancing the activity of NR is conducive to reduce the concentration of nitrate in plants. The experiments were conducted to investigate the activity of nitrate reductase in different plant tissues and the relationship between external inducing solution concentration and NR activity (NRA) in plant leaves. Six plant seedlings growing in solution culture were deprived of an external nitrogen (N) supply for 2 weeks. On selected days, three of six plant seedlings were exposed to 50mmol/L NO3- for 0, 2, 5, 8, 11h, and four of the six plant seedlings were exposed to 0, 10, 30, 50mmol/L NO3- for 2h. The NRA was determined in vivo at 538nm using spectrophotometer. The results showed that NRA increased when those plant seedlings were induced by nitrate solution. The change trends of NRA in roots and in leaves of cole, pea and tomato were different during treating time. The NRA in cole leaves was higher than that in its root and in other two plants and increased along with inducing time, but the NRA in bea and tomato was highest when the treating time was 8h and 2h, respectively. The highest NRA in leaves of three kinds of Chinese cabbages and tomato was induced by different concentrations of KNO3 solution. In tomato leaves, the highest NRA was induced by 10 - 30mmol/L KNO3 solution. In three Chinese cabbages, Brassica chinensis L. cv. AJH, XBC and KR-605, the highest NRA was induced by 10, 30, 10mmol/L KNO3 solution, respectively. The results indicated that the response manners of NRA in plants to external nitrate solutions were different. According to these results, the level of NR mRNA in plants could be enhanced by nitrate inducement. The total RNA was isolated from tomato leaves and root which induced by 30mmol/L KNO3 solution for 2h, and NR cDNA was obtained by RT-PCR using the specific primers. The fragments of PCR products were cloned and sequenced. There are 2736 base pairs in the whole cDNA fragment. The deduced protein sequence contains 911 amino acids. The NR gene can be fused to the CaMV 35S promoter, then introduced to higher plants, such as vegetables. It is hoped to decrease drastically the nitrate content of the transgenic plants.
植物中积累的过量硝酸盐会严重影响蔬菜品质，而过量摄入硝酸盐会对人体健康造成危害。同化型NADH：硝酸还原酶（NR，EC 1.6.6.1）是一种复杂的含钼蝶呤、细胞色素b(557)和FAD的蛋白质，催化高等植物利用无机氮过程中的调控和限速步骤。提高NR的活性有助于降低植物体内硝酸盐的浓度。本实验旨在研究不同植物组织中硝酸还原酶的活性以及植物叶片中外源诱导溶液浓度与NR活性（NRA）之间的关系。将6株水培生长的植物幼苗进行2周的外源氮供应剥夺处理。在选定的日子里，6株植物幼苗中的3株分别在0、2、5、8、11小时暴露于50mmol/L的NO3-中，6株植物幼苗中的4株分别在0、10、30、50mmol/L的NO3-中暴露2小时。使用分光光度计在538nm处测定体内NRA。结果表明，这些植物幼苗经硝酸盐溶液诱导后NRA增加。在处理期间，油菜、豌豆和番茄根和叶中的NRA变化趋势不同。油菜叶片中的NRA高于其根和其他两种植物中的NRA，且随诱导时间增加，而豌豆和番茄中的NRA分别在处理时间为8小时和2小时时最高。三种大白菜和番茄叶片中的最高NRA由不同浓度的KNO3溶液诱导。在番茄叶片中，10 - 30mmol/L的KNO3溶液诱导出最高NRA。在三种大白菜，即小白菜品种AJH、XBC和KR - 605中，最高NRA分别由10、30、10mmol/L的KNO3溶液诱导。结果表明，植物中NRA对外源硝酸盐溶液的响应方式不同。根据这些结果，硝酸盐诱导可提高植物中NR mRNA的水平。从经30mmol/L KNO3溶液诱导2小时的番茄叶片和根中分离总RNA，并使用特异性引物通过RT - PCR获得NR cDNA。对PCR产物片段进行克隆和测序。整个cDNA片段有2736个碱基对。推导的蛋白质序列包含911个氨基酸。NR基因可与CaMV 35S启动子融合，然后导入高等植物，如蔬菜中。有望大幅降低转基因植物中的硝酸盐含量。
