Hughes C A, Beard H S, Matthews B F
U.S. Department of Agriculture, Agricultural Research Service, Beltsville, MD 20705, USA.
Plant Mol Biol. 1997 Jan;33(2):301-11. doi: 10.1023/a:1005784202450.
Two cDNA clones (SAS1 and SAS2) encoding different isoforms of asparagine synthetase (AS; EC 6.3.5.4) were isolated. Their DNA sequences were determined and compared. The amino-terminal residues of the predicted SAS1 and SAS2 proteins were identical to those of the glutamine binding domain of AS from pea, asparagus, Arabidopsis and human, suggesting that SAS1 and SAS2 cDNAs encode the glutamine-dependent form of AS. The open reading frames of SAS1 and SAS2 encode a protein of 579 and 581 amino acids with predicted molecular weights of 65182 and 65608 Da respectively. Similarity of the deduced amino acid sequences of SAS1 and SAS2 with other known AS sequences were 92% and 93% for pea AS1; 91% and 96% for pea AS2; 88% and 91% for asparagus; 88% and 90.5% for Arabidopsis; 70.5% and 72.5% for E. coli asnB and 61% and 63% for man. A plasmid, pSAS2E, was constructed to express the soybean AS protein in Escherichia coli. Complementation experiments revealed that the soybean AS protein was functional in E. coli. Southern blot analysis indicated that the soybean AS is part of a small gene family. AS transcript was expressed in all tissues examined, but higher levels were seen in stem and root of light-grown tissue and leaves of dark-treated tissue.
分离出了两个编码天冬酰胺合成酶（AS；EC 6.3.5.4）不同同工型的cDNA克隆（SAS1和SAS2）。测定并比较了它们的DNA序列。预测的SAS1和SAS2蛋白的氨基末端残基与豌豆、芦笋、拟南芥和人类AS的谷氨酰胺结合域的残基相同，这表明SAS1和SAS2 cDNA编码谷氨酰胺依赖性形式的AS。SAS1和SAS2的开放阅读框分别编码一个由579和581个氨基酸组成的蛋白质，预测分子量分别为65182和65608 Da。SAS1和SAS2推导的氨基酸序列与其他已知AS序列的相似性，对于豌豆AS1为92%和93%；对于豌豆AS2为91%和96%；对于芦笋为88%和91%；对于拟南芥为88%和90.5%；对于大肠杆菌asnB为70.5%和72.5%；对于人类为61%和63%。构建了一个质粒pSAS2E，用于在大肠杆菌中表达大豆AS蛋白。互补实验表明大豆AS蛋白在大肠杆菌中具有功能。Southern印迹分析表明大豆AS是一个小基因家族的一部分。AS转录本在所有检测的组织中均有表达，但在光照生长组织的茎和根以及黑暗处理组织的叶中表达水平较高。
