野生大豆转录因子GsNAC20基因的分离及胁迫耐性分析

Isolation and Tolerance Analysis of GsNAC20 Gene Linked to Response to Stress in Glycine soja

NAC(NAM,ATAF1/2,CUC2)转录因子作为一类新型转录因子已成为非生物胁迫基因工程领域的研究热点。本研究以野生大豆(Glycine soja)为材料,利用酵母单杂交的方法筛选到一个能够与MYB1AT元件(核心序列为AAACCA)结合的转录因子基因,该基因与大豆NAC20(EU440353.1)基因具有99%的相似性,命名为GsNAC20。GsNAC20蛋白含有典型的NAC结构域和转录激活区。酵母试验表明,GsNAC20转录因子能够与耐逆相关顺式元件MYB1AT特异结合,但不具有自激活功能。细胞定位分析证明该基因位于细胞核中,符合转录因子的特征。GsNAC20能够响应高盐、干旱和低温胁迫,并且在根和叶中具有不同的表达模式。超量表达GsNAC20基因的拟南芥对盐胁迫的敏感性提高。以上结果表明GsNAC20参与植物非生物胁迫反应过程,该基因在非生物胁迫基因工程研究领域具有良好的理论研究和实际应用价值。

Abiotic stresses, such as salt and drought, affect plant growth, development and reduce crop yield. Isolation of a key regulatory gene linked to response to abiotic-stress and identification of the genes function are urgently needed. Glycine soja is an excellent material to isolate abiotic stress-related genes because of its high stress tolerance. Plant-specific transcription factor NAC （NAM, ATAF1/2, CUC2） proteins play essential roles in many biological processes such as development, senescence, morphogenesis, and stress signal transduction pathways. It has become a new research focus in the abiotic-stress field. Based on that, we screened a new NAC gene from Glycine soja by yeast one hybrid, which has 99% similarity with NAC20 of Glycine max （EU440353.1）, named as GsNAC20. GsNAC20 had typical NAC DNA-binding domain at the N-terminal and transcription activa-tion region at the C-terminal. It can bind to MYB1AT element （the core sequence：AAACCA） in vitro, but no transcriptional acti-vation activity in the yeast assay system, which was consistent with GmNAC20. Localization of GsNAC20 protein was analyzed by transient expression in tobacco epidermis cells and the result showed that GsNAC20 was localized in nucleus. Semi-quantitative RT-PCR showed the expression level of GsNAC20 was induced by drought, low temperature and salt stresses, but there existed difference between leaf and root in G. soja. Arabidopsis thaliana plants overexpressing GsNAC20 showed higher sensitivity under salt stress. All results showed that GsNAC20 perhaps is a new member of NAC family in G. soja, 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.