转录因子PtERF-1调控杨树抗涝性的分子机制解析

Soil flooding stress is one of the most impotant limiting factors in poplar growth of waterlogging area. In our previous study, we cloned a transcription factor PtERF-1 by RNA-seq in two full-sib poplar clones differing in flood tolerance. The gene was strongly induced by flood stress in the flood-tolerant clone, but not induced in another flood-intolerant one. Flood tolerance could be significantly increased by overexpressing the gene in Arabidopsis thaliana. On these bases, the project will primarily investigate the characteristics of PtERF-1 as a transcription factor. Its overexpression vector, RNAi vector and dominant repressing vector will be constructed and transform poplar, respectively, for its functional identification of flood resistance in poplar. RNA-Seq will be subsequently performed in these transgenic and non- transgenic poplar plants after flooding treatment to select different expressed genes. Metabolism and anatomy analysis involved these different expressed genes will be conducted. Target genes of PtERF-1 will be identified combined with analysis of yeast one-hybrid, ChIP-Seq and EMSA. In addition, yeast two-hybrid analysis will be performed to define the interaction factors of PtERF-1. The project is the extension of previous studies, which will provide important gene resource for poplar genetic improvement in flood tolerance. These results will also benefit revealing the regulation mechanism of PtERF-1 in poplar flood tolerance, and supply theory evidences for flooding stress response network in plants involved the gene.

淹水胁迫是涝渍地区杨树生长最主要的限制因子之一。前期研究中，我们通过对2个抗涝性显著不同的杨树全同胞子代的转录组分析，克隆到转录因子PtERF-1，并发现它在抗涝杨树中受淹水胁迫强烈诱导，而在不抗涝杨树中未被诱导。在拟南芥中超量表达该基因，可以显著提高抗涝性。在此基础上，本项目拟分析PtERF-1作为转录因子的基本特征。构建超量表达载体、RNAi载体和显性抑制载体转化杨树，明确PtERF-1在杨树中的抗涝功能。对淹水处理前后转基因和非转基因植株进行转录组测序，筛选差异表达基因，并对其涉及的代谢途径进行生理测定和解剖结构分析。结合酵母单杂交、ChIP-Seq、EMSA分析，确定PtERF-1的靶基因。采用酵母双杂交等方法，发掘PtERF-1的互作因子。本项目是已有研究的深入和延续，将为杨树抗涝育种提供重要的基因资源，并揭示PtERF-1的作用机制，为以它为节点的淹水胁迫应答网络提供理论依据。

以杨树PtERF-1基因为对象，围绕该基因调控杨树抗涝性的功能、转录因子基本特征、调控下游靶基因以及互作蛋白开展研究。同时，针对杨树抗涝这一性状，开展了植株不同组织部位对淹水胁迫响应的差异分析，以及不同外源化合物对杨树抗涝性影响的研究，并构建了杨树抗涝分离群体以及该群体的高密度遗传图谱，为后续杨树抗涝基因定位与抗涝分子机理研究奠定了基础。研究结果显示，PtERF-1基因在杨树的各个组织中均有表达，淹水胁迫导致其表达量发生明显变化。在细根中，淹水处理6 h后，PtERF-1基因的表达量显著上调，随后降低，并于24 h达到最低值，然后逐渐升高，于168 h后达到最高值。PtERF-1基因的启动子可诱导GUS基因在叶片、细根和茎中表达，以叶片和细根中表达量最高。抗涝无性系LS1的PtERF-1基因启动子诱导GUS基因表达量强于不抗涝无性系LS2的启动子。PtERF-1基因定位于细胞核中，具有转录激活活性，激活区位于577-1044bp区域。PtERF-1基因具有调控杨树抗涝性的功能，过量表达该基因可以提高杨树的抗涝性。PtERF-1基因调控的下游靶基因可能包括Potri.014G184200、Potri.006G017700、Potri.011G110000和Potri.001G098800等，并与其互作从而调控杨树的抗涝性。杨树不同叶位的叶片对淹水胁迫的响应存在差异。淹水胁迫下，细根中通气组织的形成方向是由主根向根尖。叶面喷施脯氨酸20 mM可提高杨树的抗涝性，增加淹水胁迫下的生物量积累。采用I-69杨×小叶杨子代构建了抗涝性差异明显的抗涝分离群体，并采用高通量GBS技术得到92097个高质量SNPs标记，用其中889个和1650个SNPs分别构成了母本I-69杨和父本小叶杨的高密度遗传图谱，该遗传图谱可用于后续该群体的数量性状定位分析。