玉米ZmWRKY20基因应答高盐胁迫的分子机制研究

Soil salinity is the main factor that can adversely affect maize growth, development and productivity. It is therefore imperative to study the molecular mechanism of salt tolerance in maize, to identify new tolerant genes, and to breed new cultivars with tolerance to salt stress. In our previous works, we characterized one salt-tolerant gene, ZmWRKY20, through screening B73 background mutant library and MutMap cloning methods. Overexpression of ZmWRKY20 remarkably reduced plant tolerance to salt stress in transgenic rice. To further elucidate the molecular mechanism underlying ZmWRKY20-mediated salt stress tolerance in maize, the following experiments will be performed: (1) Yeast two-hybrid assay and Pull down assay will be used to investigate the interaction proteins of ZmWRKY20. (2) To verify the role of ZmWRKY20 gene in salt stress response in maize, ZmWRKY20 -overexpressing plants will be generated. (3) The phytohormone content in transgenic/mutant and control plants under different conditions will be tested to explore the regulatory network administered by ZmWRKY20. (4) The high-throughput sequencing technology will be employed to identify the differentially expressed genes between transgenic/mutant and control plants, especially for these genes involved in different stress signal pathways. (5) EMSA was used to verify the target gene of ZmWRKY20 and its function also will be investigated. The findings of this study will provide important theoretical guidance and practical application value in revealing the molecular mechanism of WRKY protein involved in salt stress response and breeding new maize germplasm with improved stress tolerance.

盐害是影响玉米生长及产量的主要因素。挖掘玉米耐盐基因并利用其培育抗逆新种质是应对盐胁迫的有效途径。课题组通过筛选B73背景耐盐突变体，并结合MutMap方法克隆得到一个耐盐基因ZmWRKY20。过量表达该基因可显著降低转基因水稻的耐盐性。本研究中，为阐明ZmWRKY20调控玉米耐盐的分子机制，我们将开展以下方面的工作：（1）利用酵母双杂交等实验筛选及验证ZmWRKY20的互作蛋白；（2）构建过表达载体转化玉米，确认ZmWRKY20的耐盐功能；（3）测定转基因及突变体植株在盐处理前后的激素含量，探求ZmWRKY20在调控玉米耐盐过程中参与的信号途径；（4）利用转录组测序技术获得转基因植株、突变体植株的转录组数据，了解ZmWRKY20的过量表达及突变对玉米全基因组转录水平的影响；（5）EMSA验证ZmWRKY20靶基因及其功能确认。项目研究结果将为玉米耐盐种质创建提供新的理论指导和基因资源。

WRKY转录因子在植物盐胁迫应答过程中起到重要作用。本研究基于EMS诱变的玉米突变体库进行耐盐材料筛选，获得一份耐盐突变体。通过MutMap方法克隆得到一个盐胁迫相关基因ZmWRKY20。高盐胁迫处理后，ZmWRKY20基因的表达水平上调。组织表达模式分析表明ZmWRKY20在叶中的表达水平最高。该蛋白定位在细胞核上且在酵母中表现出转录活性。酵母单杂交、凝胶迁移实验和双荧光素酶检测报告实验证明ZmWRKY20可以和W-box序列特异性结合。ZmWRKY20在玉米中的过量表达能够降低转基因植株对盐胁迫处理的耐受性。进一步的研究证实ZmWRKY20可以与ZmWRKY115相互作用。两个蛋白质的协同作用增强了对下游靶基因ZmbZIP111的抑制作用。综合上述结果，本研究不仅为探讨ZmWRKY20参与玉米盐胁迫应答的分子机制奠定了坚实的工作基础，同时对于认识WRKY成员之间的关系提供了重要的线索。项目资助发表论文4篇，培养博士研究生1名，硕士研究生2名，3名同学都已取得博士/硕士学位。项目投入经费27万元，支出25.4124万元，各项支出基本与预算相符。剩余经费1.5876万元，剩余经费计划用于本项目研究后续支出。

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