TaABL1蛋白抗逆功能鉴定及胁迫应答调控机制研究

Research shows that the function of bZIP transcription factors in the model plant Arabidopsis thaliana is clear and plays critical roles in resisting abiotic stress. Now,however,about the function and molecular regulation mechanism of bZIP transcription factors in wheat less study is reported. In this study, based on the strategies of yeast one-hybrid and bioinformatics, a novel bZIP transcription factor gene, named TaABL1, was isolated and cloned from wheat. In order to identify gene function and the regulatory mechanism of the cis-elements in the promoter region, we constructed plant expression vectors and transferred wheat,through identifying drought-tolerant phenotype,measuring physiological indexes and transcriptomic sequencing analysis of transgenic wheat so as to study the function of downstream genes and analyze the expression differences of these genes and the logical relationship with the physiological changes, prove the adversity-resistant function of TaABL1 protein. In addition, by the analysis and prediction of TaABL1 gene promoter cis-elements, we constructed the transient expression vectors and transformed wheat callus, by histochemical staining and fluorescence quantitative analysis, finally claritied stress-responsive elements of TaABL1 promoter. Using the yeast one-hybrid system, its regulatory target proteins were screened and the combination characteristics were identified by EMSAs(Electrophoretic Mobility Shift Assays) experiments in vitro and transcription activation analysis in yeast vivo, demonstrated the stress-response regulatory molecular mechanisms of TaABL1 gene.Thus, for wheat adversity-resistance transgenic molecular breeding provided excellent candidate gene resources.

研究表明，bZIP类转录因子在模式植物拟南芥中功能研究较清楚，在调控抵御非生物逆境胁迫中起着至关重要的作用。然而目前关于小麦中的bZIP转录因子的功能及逆境应答调控机制研究报道较少。本研究基于酵母单杂系统及生物信息学策略从小麦中克隆了一个bZIP转录因子基因TaABL1，拟构建植物表达载体转化小麦，通过抗旱表型鉴定、生理指标测定及转基因小麦转录组测序分析，剖析下游基因表达差异变化和生理代谢变化之间的逻辑关系，明确TaABL1蛋白在逆境胁迫条件下抗逆性功能。此外，通过预测TaABL1基因启动子中的调控元件，构建瞬时表达载体转化小麦愈伤组织，经组织化学染色、荧光定量分析明确逆境胁迫作用元件，采用酵母单杂交系统筛选其调控靶蛋白，通过对靶蛋白和作用元件进行凝胶组织体外结合特性分析及酵母体内转录激活特性验证，阐明TaABL1基因逆境应答调调控机制。从而，为小麦抗逆转基因分子育种提供优异候选基因资源。

研究表明，bZIP类转录因子在植物抵御干旱、高盐、低温等非生物逆境胁迫中起着至关重要的作用。然而目前关于小麦中抗逆相关的bZIP转录因子功能研究及逆境应答调控机制报道较少。本研究克隆获得的TaABL1 bZIP转录因子基因，通过转基因拟南芥功能验证，明确了该基因的表达能够显著增强转基因拟南芥的抗旱、耐盐、耐低温及氧化胁迫；进一步构建了基因枪过表达载体转化小麦，通过温室筛盒及田间抗旱表型鉴定、生理指标测定分析，筛选、获得了4个T2代抗旱转基因小麦优异株系材料；通过TAIL-PCR克隆、获得了TaABL1基因2kb启动子全长序列，并对其调控元件预测分析，构建了含有ABA、干旱等不同顺式作用元件的表达载体转化小麦愈伤组织，经组织化学染色、GUS荧光定量分析明确了1个MBS干旱响应元件；构建了含有MBS作用元件的酵母报道子载体，采用酵母单杂交系统筛选、获得了其上游TaTPA46调控因子；通过对TaTPA46靶蛋白和MBS作用元件进行酵母体内转录激活验证，初步阐明了TaABL1基因逆境应答上游调控分子机制，该项目成果为今后小麦转基因分子育种提供了非常重要的理论依据和优异的抗逆基因资源。