【摘要】 [目的]本文通过对水稻甘氨酸富集基因OsGRP1的研究,从分子角度及生理遗传角度分析其在ABA诱导的抗氧化防护中的作用,并探究OsGRP1是否影响植物抗氧化防护与植物细胞壁发育。[方法]克隆基因分别构建相应重组质粒,用酵母双杂交试验（yeast two-hybrid, Y2H）研究转录因子ZFP36是否与OsGRP1蛋白水平互作,并通过GST pull-down（glutamine-S-transferase pull-down）、免疫共沉淀Co-IP（coimmunop-recipitation）、双分子荧光互补试验（bimolrcular fluorescence complementation, BiFC）辅证互作的真实性。观察ABA和H₂O₂条件下不同时间处理野生型水稻幼苗后OsGRP1转录水平表达的变化情况,鉴定OsGRP1转基因材料并测定突变体中与抗氧化防护相关的CAT和SOD活性及OsCatB基因和OsSodCc2基因的表达量,利用遗传材料分析在干旱和氧化胁迫下的耐逆性。[结果]OsGRP1基因的CDS序列为642 bp,编码214个氨基酸。通过Y2H、GST pull-down、Co-IP、BiFC证实OsGRP1与ZFP36互作的真实性。ABA和H₂O₂等处理均可诱导OsGRP1基因的表达上调。超表达OsGRP1基因可以提高抗氧化防护酶CAT和SOD活性及诱导OsCatB和OsSodCc2基因上调表达,并且OsGRP1-OE水稻在干旱和氧化胁迫下的存活率明显高于野生型水稻,而OsGRP1-KO水稻在干旱和氧化胁迫下的存活率明显低于野生型水稻,证明超表达OsGRP1基因增强了水稻对干旱胁迫和氧化胁迫的耐受性。[结论]OsGRP1参与ABA诱导的抗氧化防护途径并增强了水稻对干旱和氧化胁迫的耐性。更多还原

【Abstract】 [Objectives]The purpose of this paper was to analyze the role of rice glycine enrichment gene OsGRP1 in ABA-induced antioxidant protection from molecular and physiological genetic perspectives, and to investigate whether OsGRP1 affected plant antioxidant protection and plant cell wall development. [Methods]Genes were cloned to construct corresponding recombinant plasmids. Yeast two-hybrid（Y2 H）was used to study whether the transcription factor ZFP36 interacted with the OsGRP1 protein level, and GST pull-down（glutamine-S-transferase pull-down）,Co-IP（coimmunop-recipitation）,and bimolrcular fluorescence complementation（BiFC）supported the authenticity of the interaction. We observed the changes of OsGRP1 transcript level expression after different time treatments of wild-type rice seedlings under ABA and H₂O₂ conditions, identified OsGRP1 transgenic material and measured CAT and SOD activities related to antioxidant protection and OsCatB gene and OsSodCc2 gene expression in the mutants, and analyzed the tolerance under drought and oxidative stress using genetic material. [Results]The CDS sequence of OsGRP1 gene was 642 bp, encoding 214 amino acids. The authenticity of the interaction between OsGRP1 and ZFP36 was confirmed by Y2 H,GST pull-down, Co-IP,and BiFC. Treatments such as ABA and H₂O₂ would all induce the up-regulation of OsGRP1 gene expression. Overexpression of OsGRP1 gene increased the activity of antioxidant protective enzymes CAT and SOD and induced up-regulated expression of OsCatB gene and OsSodCc2 gene, and the survival rate of OsGRP1-OE rice under drought and oxidative stress was significantly higher than that of wild-type rice, while the survival rate of OsGRP1-KO rice under drought and oxidative stress was significantly lower than that of wild-type rice, demonstrating that overexpression of OsGRP1 gene enhanced the tolerance of rice to drought stress and oxidative stress. [Conclusions]OsGRP1 participated in the ABA-induced antioxidant defense pathway, and enhanced the tolerance of rice to drought and oxidative stress.更多还原