转GmAREB基因提高拟南芥的干旱、氧化胁迫耐性

摘要

Abiotic stresses such as drought, high-salt and low temperature severely affect the yield and quality of crops. It is reported that subfamily A of alkalescence leucine zipper transcription factors (basic leucine zipper, bZIP) mainly participated in response to ABA, drought, high salt and oxidative stresses, plays vital roles in stress signal transduction, downstream gene expression regulation and improvement of anti-adversity in plants. In this study, soybean (Glycine max) cv. Tiefeng 8 with salt tolerance was used to isolate and obtain a bZIP transcription factor gene by electronic assembly methods, which was named as GmAREB (Glycine max ABA responsive element binding protein). This gene was composed of 1 317 nucleotides and encoded 439 amino acids. Sequence analysis showed that there were four putative phosphorylation sites (C1, C2, C3, C4), a nuclear localization signal region (KVVE) and a conservative bZIP domain. Homology and phylogenetic trees displayed that CmAREB had the higher homology and more close relationships with Arabidopsis ABF2 and rice TRAB 1. To study the combining characteristics of transcription factors and DNA, we carried out electrophoretic mobility shift assay (EMSA) experiment and the results displayed that GmAREB could specifically bind the ABRE cis-element in vivo. Functional identification of stress treatments showed that the survival rate of 35S∷GmAREB transgenic plants treated by drought was higher (50％) than that (5％) of wild type. Stomatal observation displayed that the stomatal aperture of transgenic Arabidopsis (0.8 μm) was less than that of control (2.6 μm) under the drought stress for 2 h. The 35S::GmAREB transgenic plants were treated with methyl viologen (MV) to identify the oxidative stress tolerance of the GmAREB gene. Statistical analysis showed that transgenic Arabidopsis maintained higher chlorophyl content (7.3 mg g-1 FW) than that of the wild-type. RT-PCR analysis of transgenic Arabidopsis showed that GmAREB overexpression enhanced the expression of downstream stress-related target genes (AB11, AB12) and suppressed the stomatal opening-related target genes (KAT1, KAT2). Therefore, GmAREB overexpression effectively regulated the expression of downstream target genes,accelerated the stomatal closure, reduced the moisture to evaporate, decreased chlorophyll degradation, and enhanced the tolerance to drought and oxidative stresses in transgenic Arabidopsis.%以耐盐性较强的大豆(Glycine max L.)品种铁丰8号为试验材料,克隆到1个A亚族bZIP类转录因子基因,命名为GmAREB(Glycine max ABA responsive element binding protein).该基因由1 317个核苷酸组成,编码439个氨基酸残基,包括4个保守的磷酸化位点区域(C1、C2、C3和C4)、1个核定位信号区(KVVE)和1个bZIP转录因子保守域.聚类分析显示GmAREB蛋白与拟南芥ABF2、水稻OsTRAB1具有较高的同源性,并存在较近的亲缘关系.采用凝胶阻滞实验方法证明GmAREB蛋白与ABRE顺式元件具有体外结合特异性.功能分析结果表明,在干旱胁迫条件下,GmAREB转基因拟南芥的存活率(50%)显著高于野生型(5%);气孔统计分析显示转基因植株的气孔开度(0.8 μm)明显比对照开度小(2.6 μm).氧化胁迫结果显示GmAREB转基因拟南芥在甲基紫精溶液中叶绿素含量比野生型高(7.3 mg g-1 FW).转基因拟南芥RT-PCR分析表明,GmAREB基因过表达能够增强下游胁迫相关基因ABI1、ABI2的表达,抑制气孔开闭相关基因KAT1、KAT2的表达.综上所述,GmAREB基因过表达有效调控了转基因拟南芥下游靶基因表达,加速了气孔关闭,减少了水分蒸发和叶绿素降解,从而提高了转基因拟南芥对干旱、氧化胁迫耐性.