水稻叶片中脉发育基因DL2的功能验证及调控的分子机理研究

Leaf veins in plant play a key role in transporting water and photosynthates and providing mechanical support for leaf. Leaf venation pattern determines the leaf architecture which affects the light-receving and photosynthesis of plant. In our previous study a rice mutant exhibiting drooping leaf (dl2) was characterized, the physiological mechanism of midrib-less mutation was anlayzed and the candidate gene (DL2) responsible for the mutation was positionally cloned. Here the biological function of DL2 will be confirmed by complementary experiment and RNA interference. In addition, the spatio-temporal expression patterns, transcription regulation and the protein sublocalization of DL2 will be further determined. To reveal the the molecular details of DL2 regulating leaf venation, the effects of dl2 mutation on concentration and distribution of the signaling molecules in phosphatidylinositol signaling pathways, and the early details in differentiation process of leaf veins will be investigated, and also the transcriptomic, proteinomic and metabolomic changes caused by dl2 mutation will be assessed in detail. On the basis of this, combined with the data of protein interacted with DL2 protein, the signaling transduction pathways related to plant development will be primarily probed. This study will contribute to understanding of the molecular mechanism of leaf venation and vascular pattern formation in monocot species, as well as improving leaf architecture and phothsynthesis for rice and the grass family plants.

叶脉在植物生长发育过程中为叶片输送水分和营养，且叶脉发育状况直接影响株型及叶片受光率，从而影响光合效率。我们前期利用水稻无中叶脉突变体dl2研究了水稻叶片中脉发育的生理机制，克隆了水稻叶片中脉发育基因(DL2)的候选基因。本项目拟在现有基础上，采用基因功能互补及RNAi技术验证DL2的生物学功能，研究DL2的时空表达模式及其转录调控特性，分析蛋白的亚细胞定位；在确定DL2功能的基础上，研究dl2突变对三磷酸肌醇信号传导途径中重要信号分子浓度与分布的改变，及对叶脉早期发育的影响，并结合dl2突变对水稻基因转录谱、蛋白表达谱及代谢谱的影响，揭示DL2调控叶脉发育的分子机制；结合酵母双杂交技术，初步探讨DL2参与的植物生长发育信号传导途径。该研究在理论上对于深入理解单子叶植物叶脉及维管束系统发育的分子机制具有重要意义，在实践上将为水稻乃至其它禾本科作物的株型改造、提高植物光能利用率提供指导依据。

叶脉在植物生长发育过程中为叶片输送水分和营养，且叶脉发育状况直接影响植物株型及叶片受光率，从而影响光合效率。水稻是世界上最重要的粮食作物之一，也是单子叶植物的模式植物。我们以水稻无中叶脉突变体dl2为材料克隆了水稻叶片中脉发育基因(DL2)。本研究采用基因功能互补实验验证了DL2的生物学功能，研究了DL2的时空表达模式及其转录调控特性，分析了dl2突变对三磷酸肌醇信号传导途径中重要信号分子IP3和(Ca2+)cyt浓度的改变，及对生长素信号途径中生长素生物合成及生长素极性运输的影响；最后研究了dl2突变对水稻转录谱、蛋白组及代谢谱的影响。研究结果表明：DL2主要集中叶片和叶鞘表达，而且能挽救dl2突变体叶片披垂的表型，说明DL2控制水稻叶脉和维管束的发育；此外，转录组和蛋白组学研究也表明野生型和dl2突变体之间的差异表达基因和差异表达蛋白参与维管束的发育；dl2突变影响了水稻叶片中IP3和(Ca2+)cyt的浓度, 而且代谢谱研究也表明dl2突变体叶片中IP3也显著上升，说明DL2可能参与三磷酸肌醇信号及钙离子信号途径；dl2突变体中生长素生物合成及生长素极性运输基因的表达水平显著下降，并最终导致dl2突变体叶片中生长素浓度显著降低；代谢谱研究表明dl2突变体叶片IAN(IAA前体物质)浓度显著上升，说明dl2突变影响了生长素在水稻叶片中的累积，DL2可能参生长素信号途径。DL2的互作蛋白研究结果也表明， DL2可能参与生长素信号途径，而且还可能参与ABA信号途径，并参与非生物胁迫反应。该研究结果在理论上对于深入理解单子叶植物叶脉及维管束系统发育的分子机制具有重要意义，在实践上将为水稻乃至其它禾本科作物的株型改造、提高植物光能利用率提供指导依据。