花生水分胁迫下AhAREB1对AhNCED1启动子的转录调控及其必需蛋白基序作用研究

In our preliminary studies, we have cloned from peanut (Arachis hypogaea L.) the AhAREB1 gene that is characterized as a transcript factor. We have found that the ectopic expression of the gene in Arabidopsis can improve plant resistance to osmotic stress. In this research, on the basis of understanding the specific binding of the AhAREB1 protein to cis-acting elements of the promoter DNA fragments of AhNCED1 encoding a key enzyme during peanut abscisic acid (ABA) biosynthesis in vitro and subsequently the specific binding in vivo under water stress, and using transient and stable expression, we will further: (1) analyze the effects of different protein fragments of AhAREB1 on the efficiency of AhNCED1 promoter DNA binding and the activity of pAhNCED1::GUS; (2) determine the essential amino acid sections of AhAREB1 protein that affect AhNCED1 promoter DNA binding and transcriptional activity; (3) understand the transcriptional regulation model of AhNCED1 promoter by AhAREB1 under water stress, and the possible role of the essential protein motifs involved, and (4) explore the impact of water stress on the transcriptional regulation of AhNCED1 by AhAREB1. The proposed research will not only further our in-depth study on the regulation of AhNCED1 expression by AhAREB1 transcription factor, and help understand the signal transduction from the acceptance of dry signal to ABA biosynthesis and induction of gene expression in peanut, but also will provide a basis for carrying out the molecular marker-assisted breeding in peanut.

本实验室已从花生克隆了AhAREB1基因，确认其具有转录因子特性，异源表达拟南芥可提高植株渗透胁迫抗性。本研究将在明确AhAREB1蛋白与花生ABA合成关键酶AhNCED1启动子DNA各区段顺式作用元件的体外特异结合的基础上，进一步研究其体内特异结合，通过瞬时和稳定表达，分析AhAREB1不同区段对AhNCED1启动子DNA结合效率和pAhNCED1::GUS活性的影响，明确影响AhNCED1启动子DNA结合和转录活性的AhAREB1蛋白的必需氨基酸区段；认识水分胁迫下AhAREB1对AhNCED1基因启动子的转录调控模式及其必需蛋白基序的可能作用，探讨水分胁迫对AhAREB1转录调节AhNCED1产生的影响。这些工作将为我们深入研究AhAREB1转录因子对AhNCED1表达的调控、为了解花生接受干旱信号到脱落酸合成及诱导基因表达期间的信号传导提供基础，为开展花生分子标记辅助育种提供依据。

本实验室前期工作已从花生中克隆了AhAREB1基因，确认其具有转录因子特性并在拟南芥异源表达可提高植株渗透胁迫抗性。本研究在明确AhAREB1蛋白与花生ABA合成关键酶AhNCED1启动子DNA各区段顺式作用元件的体外特异结合的基础上，通过瞬时和稳定表达发现水分胁迫下AhAREB1转录因子与AhNCED1启动子DNA特异结合后负调控AhNCED1转录表达。发现AhAREB1对AhNCED1启动子DNA结合和转录激活的必需结构域为AhAREB1蛋白N端C2段。AhAREB1保守结构域C1、C2和C3的缺失不影响AhAREB1进入细胞核，CHIP结果表明AhAREB1能通过结合基因AhNCED1启动子上的第二、四个ABRE顺式元件负调控基因表达。AhAREB1蛋白诱导表达具组织特异性，仅在花生叶片与根有所表达，并随着水分胁迫程度加深明显上调。ABA可以通过延缓AhAREB1蛋白降解以增加蛋白累积，使AhAREB1蛋白迅速累积并一直保持高水平。水分胁迫下AhAREB1和AhNCED1在花生组织中呈干旱诱导，且波动表达的趋势。CHIP结果表明水分胁迫下AhAREB1对AhNCED1的调控作用更加强烈。这些工作将为我们深入研究AhAREB1转录因子对AhNCED1表达的调控，了解花生接受干旱信号到脱落酸合成及诱导基因表达期间的信号传导提供基础，为开展花生分子标记辅助育种提供依据。