杨树WRKY40基因参与抗旱的分子机制研究

WRKY transcription factors are demonstrated to be involved in regulation of drought and salt tolerance in plants. In Arabidopsis, AtWRKY40 is a down-regulator of abscisic acid (ABA) mediated signaling pathway, but its up-stream regulation has not been reported. It remains unknown whether PalWRKY40 (from Populus alba var. pyramidalis), a poplar homologue of AtWRKY40 is involved in drought tolerance and how it is regulated by the up-stream genes. In this study, we aim to explore expressions of physiological traits and related genes under drought stress through over- and down- expression of PalWRKY40 in transgenic poplars. We will further detect the up-stream regulators of PalWRKY40 in poplars via yeast one hybrid screening. Finally, we will examine interactions between the mRNA of PalWRKY40 and candidate miRNA via RACE to elucidate its post-transcription regulations. All these results not only highlight the molecular mechanism of poplars under drought stress, but also provide evidence for breeding genetically modified poplars with the high drought tolerance.

WRKY转录因子广泛参与了植物抗旱抗盐碱途径的调控，在拟南芥中证明AtWRKY40是脱落酸介导的抗旱通路的负调控蛋白，但AtWRKY40基因的上游和转录后调控机制未见报道。与拟南芥AtWRKY40基因同源的杨树同源基因(新疆杨PalWRKY40)是否参与抗旱、尤其是其上游和转录后调控机制需要深入研究。我们前期的生物信息学分析预测新疆杨PalWRKY40启动子上存在干旱响应元件。因此本项目拟通过对新疆杨PalWRKY40基因过量表达和表达下调的转基因杨树进行抗旱性状分析和相关基因表达情况检测，阐明该基因在杨树抗旱途径中的生物学功能；利用酵母单杂交文库筛选调控该基因表达的上游调控基因；通过RACE技术分析该基因与候选miRNA的相互作用关系，阐明其转录后调控分子机制。研究结果将为抗旱杨树的选育提供部分依据，具有一定的理论和实践意义。

盐胁迫是最为重要的逆境因子之一，严重影响了植物的生长发育。植物可通过激活离子外排通道，关闭气孔和清除活性氧等方式应对盐胁迫带来的负面影响，其中脱落酸（ABA）信号通路广泛参与了这些过程。然而ABA信号通路介导的盐胁迫响应过程在多年生木本植物中研究甚少。在本研究中，我们通过分析新疆杨盐胁迫前后的转录组数据，筛选到表达水平受盐胁迫和ABA处理负调控的WRKY转录因子基因，PalWRKY77，并鉴定到了其响应盐胁迫的关键启动子区域，同时还分析了PalWRKY77的亚细胞定位。过量表达PalWRKY77的转基因新疆杨对ABA不敏感，而更不抗盐。CRISPR-CAS9系统介导产生的palwrky77突变体杨树对ABA的敏感性增强，而更加抗盐。通过分析盐胁迫响应和ABA信号通路相关基因表达情况，发现盐耐受正调控基因RD26和NAC002在palwrky77突变体植株中表达水平显著提高。进一步分析发现，PalWRKY77通过结合RD26和NAC002启动子上的W盒，直接调控两基因的表达。另外，E3泛素链接酶PUB79可与PalWRKY77相互作用，并介导PalWRKY77的泛素化降解，从而解除PalWRKY77对下游靶基因的抑制作用，而PUB79的转录又受PalWRKY77的负调控。这项研究解析了PalWRKY77在杨树盐响应和ABA信号通路中的生物学功能和转录调控网络，加深对木本植物响应盐胁迫的分子调控机制的理解，为进一步选育耐盐抗旱的林木新品种提供基因资源和重要思路。