Waterhouse R N, Smyth A J, Massonneau A, Prosser I M, Clarkson D T
Department of Agricultural Sciences, University of Bristol, UK.
Plant Mol Biol. 1996 Mar;30(5):883-97. doi: 10.1007/BF00020801.
Two cDNA clones, LJAS1 and LJAS2, encoding different asparagine synthetases (AS) have been identified and sequenced and their expression in Lotus japonicus characterised. Analysis of predicted amino acid sequences indicted a high level of identity with other plant AS sequences. No other AS genes were detected in the L. japonicus genome. LJAS1 gene expression was found to be root-enhanced and lower levels of transcript were also identified in photosynthetic tissues. In contrast, LJAS2 gene expression was root-specific. These patterns of AS gene expression are different from those seen in pea. AS gene expression was monitored throughout a 16 h light/8 h dark day, under nitrate-sufficient conditions. Neither transcript showed the dark-enhanced accumulation patterns previously reported for other plant AS genes. To evaluate AS activity, the molecular dynamics of asparagine synthesis were examined in vivo using 15N-ammonium labelling. A constant rate of asparagine synthesis in the roots was observed. Asparagine was the most predominant amino-component of the xylem sap and became labelled at a slightly slower rate than the asparagine in the roots, indicating that most root asparagine was located in a cytoplasmic 'transport' pool rather than in a vacuolar 'storage' pool. The steady-state mRNA levels and the 15N-labelling data suggest that light regulation of AS gene expression is not a factor controlling N-assimilation in L. japonicus roots during stable growth in N-sufficient conditions.
已鉴定并测序了两个编码不同天冬酰胺合成酶（AS）的cDNA克隆LJAS1和LJAS2，并对它们在百脉根中的表达进行了表征。对预测氨基酸序列的分析表明，其与其他植物AS序列具有高度同一性。在百脉根基因组中未检测到其他AS基因。发现LJAS1基因在根中表达增强，在光合组织中也鉴定到较低水平的转录本。相比之下，LJAS2基因的表达具有根特异性。这些AS基因的表达模式与豌豆中的不同。在硝酸盐充足的条件下，在16小时光照/8小时黑暗的一天中监测AS基因的表达。两种转录本均未显示出先前报道的其他植物AS基因的黑暗增强积累模式。为了评估AS活性，使用15N-铵标记在体内检测了天冬酰胺合成的分子动力学。观察到根中天冬酰胺合成速率恒定。天冬酰胺是木质部汁液中最主要的氨基成分，其标记速率略低于根中的天冬酰胺，这表明大多数根中天冬酰胺位于细胞质“运输”池中，而非液泡“储存”池中。稳态mRNA水平和15N标记数据表明，在氮充足条件下稳定生长期间，AS基因表达的光调节不是控制百脉根根中氮同化的因素。
