人工改造野生大豆GsDREB2基因对植物耐盐和耐渗透胁迫能力的影响

Modification of GsDREB2 from Glycine soja increases plant tolerance to salt and osmotic stress

DREB(dehydration responsive element binding protein)转录因子是一个干旱应答元件的结合蛋白,它能特异结合启动子中含有DRE/CRT顺式元件,激活逆境诱导基因的表达,调控植物对干旱、低温、高盐、高温等胁迫的耐逆性。大量研究表明DREB转录因子在信号传导、作用机理及基因表达方面存在复杂性。为了野生大豆来源GsDREB2基因能更有效地发挥功能,人工突变该基因的负向调节结构域(negative regulatory domain,NRD,140~204),经改造命名为GsDREB2-mNRD。在酵母中比较全长基因(FLDREB2)和GsDREB2-mNRD转录激活和与DRE元件结合的能力,并验证GsDREB2-mNRD核定位情况。分别将FLDREB2和GsDREB2-mNRD转化拟南芥,通过拟南芥幼苗期盐胁迫和渗透胁迫试验,比较GsDREB2-mNRD和FLDREB2在提高植物耐盐和渗透胁迫方面的差异。结果表明,GsDREB2基因内部存在着负向调节结构域(NRD),抑制了GsDREB2转录激活功能和DRE元件结合的特性;经改造的GsDREB2基因依然能定位在细胞核;超量表达GsDREB2-mNRD基因的拟南芥耐盐和渗透胁迫能力明显强于非转基因对照,也高于FLDREB2基因超表达的拟南芥;野生大豆来源的GsDREB2基因NRD结构域的缺失可增强该基因在植物耐盐、渗透胁迫等逆境胁迫下的功能。

The DREB transcription factor is a dehydration responsive element（DRE）-binding protein that specifically interacts with the DRE/CRT（C-repeat）cis-acting element in the promoter region of stress-inducible genes.This interaction controls the expression of many stress-inducible genes in plants and can increase plants＇ tolerance to drought,low temperature,high salt,and heat.Many studies have reported on the complexity of DREB signal transduction,action,and expression.To explore the role of the DREB2 gene in Glycine soja（GsDREB2）,the nucleotides encoding the negative regulatory domain（NRD,amino acid residues 140-204）were deleted fromGsDREB2.The transcriptional activation and DRE element-binding capacity were compared between proteins encoded by the full-length gene（FLDREB2）and the truncated gene GsDREB2-mNRD in yeast.The nuclear localization capacity of GsDREB2-mNRD was also evaluated.FLDREB2 and GsDREB2-mNRD were each transferred into Arabidopsis.After screening and testing,the phenotype of the transgenic Arabidopsislines was evaluated,and their stress tolerance level was determined by measuring their germination rate and fresh weight under salt and osmotic treatments.The results indicated that the amino acid region 140-204 negatively controls the transcriptional activation ability and DNA-binding capacity of DREB2.Both GsDREB2-mNRD and FLDREB2 were located in the nucleus.Transgenic Arabidopsis overexpressing GsDREB2-mNRD showed increased tolerance to salt and osmotic stress.The transgenic plants overexpressing GsDREB2-mNRDshowed a higher germination rate and fresh weight than those of plants expressing FLGsDREB.These results demonstrate that GsDREB2-mNRD,which encodes a mutated DREB2,can enhance plant tolerance to salt and osmotic stress.