野生大豆对非生物胁迫具有优良的抗性,是研究胁迫机制、挖掘抗性基因的理想材料。以耐盐野生大豆为试材,利用前期获得的盐胁迫基因芯片杂交结果和EST数据库,筛选出1个响应胁迫的3'-EST,序列分析表明,该EST编码膜联蛋白(annexin,ANN)。通过改进的5'-RACE获得了该基因的完整编码区,翻译产物与拟南芥膜联蛋白的相似性为57%,将该基因命名为GsANN。GsANN基因的翻译产物具有膜联蛋白家族特征性的结构域,无强烈的亲水/疏水性,无信号肽,无跨膜结构域,与已报道的植物ANN蛋白一致。亚细胞定位结果表明GsANN蛋白在细胞内聚集于质膜附近。GsANN基因在野生大豆叶中受盐及干旱胁迫的诱导,超量表达GsANN基因的拟南芥对盐胁迫和干旱胁迫的敏感性提高,揭示了该基因与植物抗性间的关系。
Abiotic stresses, such as salt and drought, affect plant growth, development and reduce crop yield. Glycine soja is an excellent material to clone abiotic stresses related genes because of its high stress tolerance. In previous work, an EST database was constructed using a salt-tolerant variety of G. soja, and a gene expression profile was also obtained by microarray hybridiza- tion. Expression of all G. soja ESTs was analyzed, and one 3'-EST was found to be induced by stress, representing an annexin (ANN) gene, a widely-existing super family which has been found to respond to some kinds of abiotic stresses and plays roles in many important cellular processes including membrane formation, regulation of ion channels, signal transduction and so on. Complete coding sequence of this annexin gene was obtaind with developed 5'-RACE and named as GsANN. Translation product of GsANN had 57% similarity with ANN4 of Arabidopsis thaliana. GsANN had three ANN domains, with moderate hydrophobicity/hydrophilicity, and no signal peptide or transmembrane segment, which was consistent with other ANNs of other plants. Localization of GsANN protein was analyzed by transient expression in tobacco epidermis cells and the result showed that GsANN localized around plasma membrane unevenly and might form oligomers. Semi-quantitative RT-PCR showed the expression level of GsANN was induced by drought and salt stresses in G. soja leaf. Arabidopsis thaliana plants overexpressing GsANN showed lower survival rate and higher sensitivity under both salt and drought stresses. GsANN is a novel ANN gene and is closely related to salt and drought stresses, so it can either be used as a new resource in gene engineering on stress tolerance or be further studied to provide more information for the researches on the mechanism of stress tolerance in plant.