玉米ZmDi19-5转录因子调控干旱胁迫应答的分子机制

Drought stress is the main factor that affecting the growth and development of maize, and it is one of the most effective measures to resist drought stress through identification of drought resistance genes, and further used to cultivate new stress-tolerance germplasm. Di19 transcription factor play an important role in the abiotic stress responses in plants, but its regulatory mechanism is poorly understood in maize. Based on our previous study, a Di19 family gene, ZmDi19-5, was identified from maize. Overexpression transgenic maize was obtained, and the function in drought response was also demonstrated. In this study, the core region of ZmDi19-5 promoter involved in drought response will be identified, and further used to identify upstream regulator by yeast one-hybrid. The physiological and biochemical mechanism of ZmDi19-5 will be demonstrated in regulating drought stress tolerance through analyzing the stress-related physiological and biochemical indexes of transgenic lines and mutants, and transcriptome sequencing and chromatin immunoprecipitation sequencing will be used to identify downstream genes. The interaction proteins of ZmDi19-5 will be identified to reveal the interaction mechanism by yeast two-hybrid, bimolecular fluorescence complementation and co-immunoprecipitation methods. Based on these results, the molecular mechanism of ZmDi19-5 will be elucidated in regulation of drought response in maize.

干旱胁迫是影响玉米生长和发育的主要逆境因子，挖掘抗旱基因并利用其培育抗逆新品种是应对干旱最为有效的途径之一。Di19类型转录因子在干旱响应过程中具有重要作用，但对其在玉米中的调控机制并不清楚。课题组通过前期研究鉴定了一个受干旱胁迫显著诱导表达的Di19家族基因ZmDi19-5，获得了过量表达该基因的转基因玉米植株并初步明确了其抗旱功能。本研究拟通过筛选ZmDi19-5响应干旱胁迫的关键启动子区段，然后利用其筛选酵母单杂交文库鉴定上游调控因子；通过对转基因玉米和突变体植株的耐旱性与逆境相关生理生化指标进行分析，明确ZmDi19-5参与胁迫应答的生理生化机制，并利用转录组测序和染色质免疫共沉淀测序鉴定其下游调控基因；同时，利用酵母双杂交、双分子荧光互补和免疫共沉淀技术鉴定ZmDi19-5的互作蛋白，揭示其互作调控机制。通过以上研究，从而阐明ZmDi19-5调控玉米干旱胁迫应答的分子机制。

Di19转录因子在植物生长发育、非生物胁迫应答等生物学过程中具有重要的作用，本项目基于前期研究，对干旱胁迫响应基因ZmDi19-5的生物学功能和分子调控机制开展了研究。通过生物信息学分析鉴定了7个玉米Di19蛋白，序列结构及进化分析揭示了Di19蛋白的保守进化特征，ZmDi19-5与已知功能的非生物胁迫响应基因进化关系较近；明确了玉米ZmDi19-5基因启动子具有响应干旱胁迫的表达驱动活性，其-650bp至-200bp为核心活性区域，并利用酵母单杂交鉴定了叶绿素结合蛋白、水通道蛋白、NAC转录因子等5个关键上游调控因子；创制了ZmDi19-5过量表达和基因编辑转基因玉米植株，与野生型植株相比，ZmDi19-5过量表达植株提高了对干旱的耐受性，基因编辑植株降低了对干旱的耐受性，进一步结合逆境相关指标分析，明晰了ZmDi19-5在干旱胁迫响应中的生物学功能以及生理生化机制。利用转录组测序对转基因与野生型植株的基因表达进行了分析，鉴定了多个与非生物胁迫响应以及胁迫信号传导等相关的差异表达基因，进一步开展了ChIP-Seq分析并结合转录组测序进行联合分析，筛选了ZmDi19-5调控的下游蛋白；通过酵母双杂交鉴定了ZmDi19-5重要互作蛋白并利用BiFC和pull-down等进行了互作分析，筛选了其参与互作的结构区域，揭示了ZmDi19-5与互作蛋白的互作机制。通过本项目研究，阐明了ZmDi19-5调控玉米干旱胁迫应答的分子机制，为通过分子育种创制抗旱玉米新种质提供了优良的基因资源。