WRKY转录因子调控小麦叶片衰老的分子机制研究

Senescence, the last stage of leaf development, is a complex process regulated by multiple genetical and biochemical pathways. The initiation and process of senescence are controlled by an array of external and internal factors. Leaf senescence is happening with wheat grain filling simultaneously at late development stage. Precocious leaf senescence seriously affects wheat yield and grain quality, while extended leaf functional period and delayed leaf senescence can significantly improve yield and quality traits. Two senescence-associated WRKY transcription factors, TaWRKYA and TaWRKYB were identified in our previous study. They are localized in nucleus, and showed transcription activation activity in yeast cells. TaWRKYA and TaWRKYB were up-regulated during natural- and dark induced- leaf senescence process. Over-expression of these two genes caused precocious leaf senescence in Arabidopsis. In this study, their transcriptome changes under various senescence signal induction will be detected. DNA binding sites and their interaction proteins will be analyzed. We will use over-expression transgenic plants, CRISPR/Cas9 transgenic plants, and wild-type plants to analyze the phenotypes. The senescence-associated signal transduction and regulation pathways will be illustrated. The results of this project will help us to understand function of these transcription factors on regulation of wheat leaf senescence, to elucidate the molecular mechanisms of leaf senescence. This study may also contribute to efficient manipulation in wheat breeding and genetic improvement.

衰老是叶片发育的最后阶段，其启动及进程调控受诸多内外因素影响。小麦生长后期叶片衰老与籽粒形成和灌浆成熟同步进行，叶片早衰严重影响产量和品质，而延长叶片功能期、延缓早衰则显著改善主要产量品质性状。本课题组鉴定出的两个衰老相关转录因子TaWRKYA和TaWRKYB定位于细胞核中，具有转录激活活性。在叶片自然衰老和黑暗诱导的衰老过程中上调表达，它们过表达导致拟南芥叶片提前衰老。本项目拟通过衰老信号诱导下的基因表达水平分析、靶基因及调控网络分析、过表达和CRISPR/Cas9转基因材料表型分析，揭示WRKY介导的衰老信号途径、靶基因作用位点，探讨其调控叶片衰老的分子机理。本项目预期研究结果将揭示WRKY参与小麦叶片衰老调控的分子机理，为小麦育种和遗传改良提供重要的理论依据。

衰老是叶片发育的最后阶段，其启动及进程调控受诸多内外因素影响。转录因子在叶片衰老启动、进程调控和信号转导过程中发挥重要作用。小麦生长后期叶片衰老与籽粒形成和灌浆成熟同步进行，叶片早衰严重影响产量和品质，而延长叶片功能期、延缓早衰则显著改善主要产量品质性状。本课题组鉴定出的两个衰老相关转录因子TaWRKYA和TaWRKYB定位于小麦细胞核中，具有转录激活活性。在小麦开花灌浆阶段，与幼穗、茎段、根等相比，它们在旗叶中具有高水平的表达。在叶片自然衰老和黑暗诱导的衰老过程中上调表达，在ABA、MeJA、SA等激素处理下，表达水平明显提高。将TaWRKYA和TaWRKYB基因过表达在拟南芥和二穗短柄草中，转基因植株表现出提前衰老的表型，叶绿素含量、离子渗透值、ROS积累等生理生化指标分析，和SAG12、SEN4、RBCS等衰老标记基因的qRT-PCR分子水平分析结果暗示TaWRKYA，TaWRKYB基因在叶片衰老中发挥正调控功能。在小麦中，我们建立和优化了BSMV介导的VIGS沉默体系，转基因小麦叶片的衰老进程得到了显著延缓。过表达转基因材料在ABA、MeJA、SA等激素处理下，叶片衰老进程显著加速。这些激素信号通路中关键基因的表达水平发生明显变化，表明TaWRKYA，TaWRKYB基因可能参与这些激素的信号转导过程。我们正在利用Chip-PCR、EMSA、BiFC等方法进行验证。研究结果将明确TaWRKYA，TaWRKYB基因所介导的衰老信号途径，为揭示WRKY调控小麦叶片衰老的分子机理奠定基础，为基于叶片衰老调控的小麦分子育种提供功能明确的具有自主知识产权的基因源和材料。