谷子抗旱相关转录因子SiAREB2的功能研究

Foxtail millet originated from China is our food crop, which grows mainly in arid and semiarid regions. So far, the molecular mechanisms of foxtail millet resistant to drought have not been clearly defined. We cloned an AREB-type transcription factor from foxtail millet and named SiAREB2, whose transcript level was up-regulated in response to ABA treatment and drought stress. Our preliminary experiments showed that SiAREB2 possessed some features of transcription factor, such as location in the nucleus and transcriptional activation. The heterologous expression of SiAREB2 in Arabidposis enhanced the tolerance to drought. In this project we will further obtain the SiAREB2-overexpressed and knock down transgenic plants through the agrobacterium transformation, and then compared and analyzed the phenotypes, physiological indexes and transcript levels of drought relative genes among the transgenic and the wide type foxtail millet under drought stress. The transcriptional activity sites of SiAREB2 in the foxtail millet protoplasts and tobacco will be studied using the effector-reporter system. The interacting proteins with SiAREB2 in foxtail millet will be screened and identified by yeast two-hybrid system and bimolecular fluorescence complementation system (BiFC). Our study will provide insights into the function of SiAREB2 and the regulation modes of SiAREB2 in foxtail millet, and will be helpful for us to better understand the working mechanism of foxtail millet resistant to drought as well as provide theoretical basis and gene resources for molecular breeding.

谷子是原产于我国，适宜在干旱和半干旱地区种植的粮食作物。目前为止，谷子抗旱的分子机理研究报道较少。我们从谷子中新克隆了一个响应外源ABA信号并受干旱胁迫诱导的AREB类转录因子基因命名为SiAREB2。前期实验显示，SiAREB2具有核定位、转录激活活性等转录因子的性质，拟南芥中异源表达SiAREB2能增强转基因植株的耐旱性。在此基础上，本项目拟采用农杆菌转化法获得SiAREB2超表达和干扰表达转基因植株，对转基因和野生型植株干旱胁迫下的表型，生理指标和抗旱相关基因转录水平进行比较；利用effector-reporter系统，在谷子原生质体和烟草中鉴定SiAREB2转录活性位点；通过酵母双杂和双分子荧光互补实验筛选鉴定谷子中与SiAREB2互作蛋白。研究结果将明确SiAREB2的功能，探索SiAREB2的调控模式，对解析谷子抗旱分子机制有重要科学意义，同时为分子育种提供理论基础和基因资源。

谷子是原产于我国适宜在干旱和半干旱地区种植的粮食作物，谷子对干旱胁迫具有良好的耐受能力。目前为止，谷子抗旱的分子机理研究报道较少。我们从谷子中克隆了一个响应外源ABA信号并受干旱胁迫诱导的AREB类转录因子基因命名为SiAREB2。SiAREB2具有核定位、转录激活活性等转录因子的性质，拟南芥中异源表达SiAREB2增强转基因植株的耐旱性。通过农杆菌转化获得SiAREB2超表达和基因敲除（利用CRISPR-Cas9系统）的转基因植株，对转基因和野生型植株干旱胁迫下的表型分析发现，与野生型植株相比超表达植株具有较强的耐旱性，而基因敲除植株抗旱性较差。生理指标分析发现在干旱胁迫下，超表达植株中可溶性糖含量和脯氨酸含量要高于野生型和基因敲除植株。转录组分析显示正常生长条件下胁迫相关基因在超表达植株与野生型和基因敲除植株中转录水平差异不明显，但干旱胁迫下超表达植株中许多与干旱胁迫应答相关基因包括LEA蛋白基因，热激蛋白基因，PP2C类基因和一些转录因子基因的转录水平与野生型和突变体材料相比明显升高。利用外源ABA处理转基因和野生型谷子后对上述部分基因的转录水平进行分析发现超表达和野生型植株中候选基因的转录水平升高明显，而基因敲除植株中胁迫相关候选基因转录水平变化不明显。另外，通过酵母双杂交筛选胁迫条件下的谷子cDNA文库发现，一个与磷酸化修饰相关基因和一个转录因子基因能与SiAREB2基因互作，双分子荧光互补实验和Pull-down实验结果进一步显示这两个基因与SiAREB2互作。这些结果表明SiAREB2基因是在ABA信号通路下，通过调控谷子中抵御干旱胁迫基因的表达来增强谷子抗旱性的功能。同时SiAREB2行驶功能可能需要经过胁迫诱导的蛋白磷酸化修饰来实现。本研究结果明确了SiAREB2的功能，探索SiAREB2的调控模式，对解析谷子抗旱分子机制有重要科学意义，同时为分子育种提供理论基础和基因资源。