拟南芥WRKY71转录因子调控叶片衰老的分子机制研究

Leaf senescence is a complex and well-regulated process which refers to the terminal phase of plant development. It is controlled by growth and development signal and environmental cues, and closely related to the yield and quality of crops and horticultural crops. Research on the molecular mechanism of leaf senescence is of important theoretical significance and practical value. Some WRKY transcription factors have been revealed to regulate leaf senescence in Arabidopsis. However, their target genes and molecular regulation network is not clear yet. In recent, we found that WRKY71 over-expression line showed precocious leaf senescence compared with Col-0, while that of the knock-out line was late. In addition, WRKY71 was strongly induced in the senescence leaf. On this basis, this project aims to explore the biology function of WRKY71 in the regulation of leaf senescence, use RNA-seq, ChIP-seq and EMSA to identify its downstream target genes involved in leaf senescence and molecular regulation network, determine the relationship between WRKY71 and its targets by crossing assay, and reveal the molecular mechanism of WRKY71 in regulating leaf senescence. The findings of this project will provide theoretical basis for revealing the molecular network of WRKY transcription factors in regulating leaf senescence, and improving the yield and quality of crops and horticultural crops.

叶片衰老是植物发育最后阶段的一个复杂而又有序调控的过程，受到生长发育信号和外界环境因素的调控，其与农作物和园艺作物的产量和品质密切相关。研究叶片衰老的分子机制具有重要的理论意义和实用价值。部分拟南芥WRKY转录因子被报道参与调控叶片衰老，而其下游靶基因和分子调控网络尚不清晰。我们前期研究发现WRKY71转录因子过表达系叶片相对于野生型Col-0早衰，而敲除系则相对较晚，且WRKY71在衰老叶片中显著上调表达。在此基础上，本项目拟利用上述材料分析WRKY71在叶片衰老中的生物学功能，通过RNA-seq、ChIP-seq和EMSA等技术鉴定其参与叶片衰老的下游靶基因及分子调控网络，并利用杂交互补确定其与靶基因之间的调控关系，解析WRKY71调控叶片衰老的分子机制，为进一步阐明WRKY转录因子家族调控叶片衰老的分子调控网络提供理论依据，为提高我国农作物产量、提升园艺作物品质奠定理论基础。

叶片衰老是植物发育最后阶段的一个复杂而又有序调控的过程，受到生长发育信号和外界环境因素的调控。本研究发现拟南芥WRKY71转录因子过表达系WRKY71-1D叶片相对于野生型Col-0早衰，而敲除系wrky71-1则相对较晚。进一步研究发现，WRKY71在衰老的叶片中表达明显，而且受到ACC、干旱胁迫、ABA、黑暗处理、双氧水和SA的诱导表达。在ACC、干旱胁迫和SA处理条件下，WRKY71-1D的叶片衰老明显早于Col-0，而wrky71-1则相反。转录组分析发现许多衰老相关基因（尤其是乙烯相关基因）在WRKY71的突变体中表达发生明显变化。实时定量PCR分析表明衰老相关基因SAG12、SAG13、SAG113、SEN1和SEN4，以及乙烯相关基因EIN2、EIN3和ACS2受WRKY71明显上调表达，而WRKY71则在乙烯信号途径突变体ein2-1中表达受到抑制，表明WRKY71参与到乙烯信号通路调控叶片的衰老。进一步EMSA和ChIP实验分析表明WRKY71可以结合ACS2的启动子，说明WRKY71可以直接调控乙烯合成。这些数据表明WRKY71与乙烯通路形成正反馈调控环参与到叶片衰老调控。综上，WRKY71能够介导多种因子参与调控叶片的衰老。