bHLH转录因子在花生干旱胁迫响应中的功能及其作用机制分析

Drought is one of the main factors that limit the growth of peanut (Arachis hypogaea L.) in China. The basic of developing drought-stress-resistant peanut varieties is to deeply explore molecular resistance mechanisms and genes in peanut. Basic helix-loop-helix (bHLH) is the most extensive class of transcription factors in plants, which can regulate gene expression through interaction with specific motif in target genes. bHLH transcription factor is not only universally involved in plant growth and metabolism, but also plays an important role in plants under abiotic stress. In addition, its mechanism and functional characterization is complex when under different stress. Comparing with other model plants, less deep work has been done to research the bHLH transcription factors in peanut, especially the bHLHs which are response to drought. In previous work, five bHLH TFs that response to drought and salt stress were achieved by RNA-Seq. Based on these works, this study plans to analyze promoters of bHLH TFs and identify core functional elements of promoters through a series deletion of the promoter sequences. Then, through the transgenic plants and virus-induced gene silencing (VIGS) method, the mechanism and functional characterization of these bHLH TFs will be obtained. Meanwhile, based on the results of Chromatin Immunoprecipitation-Sequence、Yeast one-hybrid and Electrophoretic Mobility Shift Assay, this study will also investigate the target gene and interaction sites of bHLH TFs. This work can provide a comprehensive understanding on this TFs family and will accelerate the breeding of peanut varieties which are resistant to drought.

干旱是制约我国花生产业发展的重要因素。深入研究花生分子抗旱机制，挖掘花生抗旱基因是利用分子生物学技术加速培育抗旱花生新品种的重要前提。bHLH转录因子家族在植物中广泛存在，参与调控植物生长和发育。研究表明，该家族同时参与植物逆境胁迫调控，功能与作用机制也具有复杂性。与其他模式植物相比，研究人员对于花生bHLH转录因子，尤其是抗旱相关bHLH转录因子了解较少。本项目拟在前期花生抗旱耐盐转录因子分析，首次挖掘花生5个bHLH转录因子并进行初步研究的基础上，克隆分析其启动子序列，利用片段缺失试验，获得启动子核心区；通过转基因、病毒诱导的基因沉默等方法对其功能和相关作用机理进行深入研究；同时，综合染色质免疫共沉淀-高通量测序、酵母单杂交和电泳迁移率检测的结果，筛选目标bHLH转录因子的靶基因及其在靶基因启动子上的作用区域。本研究将极大丰富对于花生bHLH转录因子的认识，对花生抗性育种具有重要意义。

bHLH（basic Helix–Loop–Helix）转录因子家族广泛参与调控植物生长和发育。研究表明，该家族同时参与植物逆境胁迫调控，而功能与作用机制也具有复杂性。为明确前期筛选的花生bHLH转录因子的功能与作用机制，团队构建了基因的瞬时表达载体，结果表明花生AhbHLH121与AhbHLH112均定位于细胞核。自激活活性实验证明，这两个基因具有转录自激活活性。表达模式分析表明，两个基因正常条件下在根、茎、叶组织均有表达。胁迫处理后，两个基因的表达模式不同。其中，施加干旱与盐胁迫后，叶部组织的AhbHLH121均在48小时内相对表达量升高；施加干旱胁迫后，叶部组织的AhbHLH112在12小时后趋于平稳；根部组织相对表达量在48小时内相对表达量显著升高；在茎部组织，18小时后相对表达量恢复到6小时的水平。基因超表达实验与病毒介导的基因沉默实验证明，AhbHLH121具有提高植株抗旱耐盐能力，AhbHLH112只在超表达时具有提高植株抗旱能力。酵母单杂交技术、电泳凝胶迁移技术与双荧光素酶实验表明，AhbHLH121可直接结合抗氧化酶基因启动子，调控基因表达，进而提高植株抗逆性。酵母单杂交技术初步证实AhbHLH112可直接结合POD基因启动子，从而提高植株抗旱性。基因超表达实验同时验证了AhbHLH10提高植株抗旱性、AhbHLH18提高植株耐盐性的功能。本研究丰富了对于花生bHLH转录因子的认识，对筛选花生抗性种质，培育抗性新品种具有重要意义。