【摘要】 天冬酰胺合成酶B(asparagine synthetase,AS-B;EC 6.3.5.4)参与植物氮同化过程的催化与调节。在构建的盐胁迫下桑树(Morus L.)抑制消减杂交(SSH)文库中发现一个编码天冬酰胺合成酶的基因片段,采用cDNA末端快速扩增(RACE)方法获得2 061 bp的全长序列,完整的开放读码框(ORF)长1 761 bp,编码586个氨基酸,预测蛋白分子质量65.66 kD,将该基因命名为桑树天冬酰胺合成酶基因(MaAS,GenBank登录号:HQ025955)。序列分析显示,MaAS编码的蛋白包括2个保守的功能域(谷氨酰胺-氨基转移结构域和合成酶结构域),与其它植物天冬酰胺合成酶的氨基酸序列相似性在75%以上。将MaAS基因开放读码框连接到pET28a(+)表达载体中,转化大肠杆菌BL21后,诱导表达的MaAS重组蛋白分子质量大小与预测的分子质量一致。采用邻接法构建的桑树和其它植物基于MaAS蛋白氨基酸序列的系统进化树显示:桑树、葡萄(Vitis vinif-era)、毛果杨(Populus trichocarpa)、蓖麻(Ricinus communis)聚为一类,亲缘关系较近。半定量RT-PCR分析表明,MaAS基因在桑树不同部位的转录水平存在明显差异,在盛花期的雌花中转录水平最高,尚未木质化的幼根和成熟桑椹中的转录水平较高,幼茎的茎皮和木质部中的转录水平较低,嫩叶中的转录水平很低,腋芽中的转录水平最低。更多还原

【Abstract】 Asparagine synthetase B(AS-B,EC 6.3.5.4) participates in catalysis and regulation during nitrogen assimilation of plant.In suppression subtractive hybridization cDNA library from mulberry under salt stress,a cDNA sequence was identified as a part of asparagine synthetase gene.Its full-length cDNA was obtained by rapid amplification of cDNA ends(RACE).It is 2 061 bp long and has a complete open reading frame(ORF) of 1 761 bp,which encodes a protein of 586 amino acid residues with a predicted molecular weight of 65.66 kD.The gene was designated as mulberry asparagine synthetase gene(MaAS,GenBank accession No.HQ025955).The deduced MaAS protein contained two conserved domains(glutamine-amide transfer domain and synthetase domain).Its amino acid sequence shares more than 75%identities with AS from other plant species.The complete coding sequence of MaAS was cloned into an expression vector pET28a(+) and expressed in Escherichia coli BL21.The expressed protein had a molecular weight of about 66 kD which was close to the deduced molecular weight of MaAS.A phylogenetic analysis revealed that mulberry had close relationships with Vitis vinifera,Populus trichocarpa and Ricinus communis.Semi-quantitative RT-PCR analysis showed that the MaAS transcription level was obviously different in various parts of mulberry.The transcription levels were the highest in pistillate flower during flowering stage,relatively higher in non-woody radicle and mature mulberry fruit,relatively lower in bark and xylem of tender stems,very low in young leaf,and the lowest in axillary bud.更多还原