一个新的花生磷脂酶D在干旱胁迫响应中的功能与作用机制研究

Phospholipase D (PLD) is a kind of important enzymes in signal transduction of biological membrane and plays a crucial role in responding to drought stress in plant. Some evidences demonstrated the specificity of PLDs in signal transduction with plant species and cellular processes. In our previous studies, a novel PLD gene, AhPLDα3, was isolated from peanut. The gene expression of AhPLDα3 was strongly stimulated by water deficit. More importantly, AhPLDα3 presented a remarkable stability of expression in conditions of progressive drought stress in peanut. Therefore, AhPLDα3 could be greatly important for peanut to respond to drought stress. However, the concrete function of AhPLDα3 has not been reported up to now. In this study, the temporal and spatial expression patterns of AhPLDα3 will be investigated firstly. Furthermore, analyses of plants over-expressing AhPLDα3 gene and silencing endogenous AhPLDα3 by RNAi will provide insights into the function by which this gene is involved in plant drought stress responses. In order to explore the physiological and biochemical mechanisms of AhPLDα3 response to drought, the relationship of stomata, plasma membrane and the essential elements in the signal transduction pathway of ABA induced stomata movement under water stress or treatment of exogenous ABA will be studied. On the other hand, to detect the molecular mechanism of AhPLDα3 in plant response to drought stress, verification and identification of valuable differentially expressed genes derived from leaf transcriptome using microarray and expression profiling analyses of AhPLDα3-deficient and over-expressing plants will be used to explore the pathways of AhPLDα3 involved in the drought signal transduction. All research findings about the AhPLDα3 in our present study will facilitate to provide the systemic theoretical support for explaining the mechanisms of signal transduction of drought stress in plant and to provide a theoretical basis for using molecular breeding approaches in improving drought tolerance and water use efficiency. Additionally, AhPLDα3 from peanut will provide an additional candidate gene for drought-tolerant crops improvement.

磷脂酶D（PLD）是一类重要的跨膜信号转导酶，在植物响应干旱胁迫中有重要作用。不同的PLD干旱应答机制截然不同。前期研究中，我们在花生中发现一个新的PLD基因AhPLDα3，可被干旱胁迫诱导并在干旱下持续稳定表达，推测其在干旱胁迫应答中起重要作用，具体功能尚未见报道。因此，本项目拟探明AhPLDα3的时空表达特性；采用过量表达和RNAi干扰技术获得转基因植物，明确其在干旱胁迫应答中的功能；通过分析在干旱或ABA诱导下，AhPLDα3过量表达和抑制后对气孔、细胞膜及ABA介导的干旱胁迫信号因子的影响，探明其响应干旱胁迫的生理生化机制；通过转基因植物芯片表达谱分析，明确AhPLDα3参与干旱信号转导的途径，解析其响应干旱胁迫的分子机制。本研究将为阐明干旱胁迫信号转导机制提供更系统的理论支持，为分子育种提高作物抗旱性提供理论依据和基因资源。

磷脂酶D（PLD）参与植物多个生理过程，尤其在逆境胁迫响应和信号转导中发挥重要调节作用。花生是耐旱性较强的油料作物，克隆花生PLD基因并探明其在干旱胁迫响应中的生物学功能，对作物抗旱性遗传改良具有潜在理论意义和应用价值。前期研究中，我们发现一个新的花生PLD基因AhPLDα3，可被干旱诱导上调表达，推测其在干旱胁迫应答中起重要作用，具体功能尚未见报道。本项目拟探明AhPLDα3的时空表达特性，明确其在干旱胁迫应答中的功能，探明其响应干旱胁迫的生理生化机制，进而明确AhPLDα3参与干旱信号转导的途径，解析响应干旱胁迫的分子机制。主要研究结果如下：（1）克隆获得了花生PLDα家族基因3个，它们均具有PLD家族的保守结构域和功能位点，其中在同源基因中，AhPLDα3的序列特异、系统进化差异较大，属PLDα家族新成员。（2）3个基因的表达特征存在明显差异，AhPLDα3呈组成型表达模式，其转录本在花生根、叶片和果针中大量积累，AhPLDα3可被干旱、ABA、盐等诱导上调表达。（3）AhPLDα3参与干旱胁迫ABA信号传导通路，ABA和H2O2可明显诱导AhPLDα3的转录活性，Ca2+信使、NO、NADPH等也参与了ABA激活AhPLDα3转录水平增加的过程。（4）拟南芥中过量表达AhPLDα3基因可明显增强转基因植株的耐旱性。在干旱胁迫下，转基因植株的叶片相对含水量和脯氨酸含量显著高于野生型植株，细胞质离子外渗率和叶片丙二醛含量比野生型显著降低。（5）干旱胁迫条件下，AhPLDα3基因过量表达可明显提高胁迫相关基因（RAB18、RD22、RD29A和RD29B）的转录水平，ABA诱导相关基因（NCED3和ABI1）也被上调表达。AhPLDα3基因过表达对转基因植株的生长发育未产生明显不良影响，所以AhPLDα3是改良作物抗旱性的优异候选基因。本研究为系统解析花生干旱胁迫信号转导机制提供了理论支持，为分子育种提高作物抗旱性提供了新的基因资源。本研究发表学术论文7篇，其中SCI收录论文2篇，国际学术会议论文摘要1篇，国内核心期刊论文3篇，培养博士研究生1名。