苜蓿复叶初级形态建成关键调控因子PPF2的分子机制研究及在苜蓿品质改良中的应用

Alfalfa (Medicago sativa) is often referred to as "the queen of forage crops" because it is highly productive, drought tolerant and provides high quality forage over a long period of time. Since alfalfa leaves have significantly higher nutritive value than stems, the leaf/stem ratio could be the valuable criteria in alfalfa molecular breeding. Therefore, one approach to improve alfalfa quality can be to develop cultivars with high leaf/stem ratio. In this project, we isolated and characterized the loss-of-function mutants of PINNATE PENTAFOLIATA2 (PPF2) that exhibits pinnate-like compound leaves with five leaflets in Medicago truncatula. Genetic analysis suggests that PPF2 plays a critical role in the primary morphogenesis (PM) stage of compound leaves. Further investigations revealed that PPF2 encoding one BEL1-LIKE HOMEODOMAIN (BLH) protein interacted with the members of MtKNOXI proteins and inhibited the activities of MtKNOX7 in protein level. Additionally, SGL1/MtUFO module is involved in PM of compound leaf regulated by PPF2. This project will investigate the regulation network, including PPF2 and two key regulatory elements of compound leaf development, MtKNOXI and SGL1/MtUFO. It will shed the light on the compound leaf patterning in Medicago, and establish foundation to reveal the mechanism of compound leaf development. In addition, we will apply these key regulators for the molecular design of leaf patterning in alfalfa and provide the insights for the forage quality improvement of alfalfa.

紫花苜蓿由于产量大、耐旱性强等特点，被誉为“牧草之王”。由于苜蓿叶片的蛋白含量要显著高于茎，所以叶茎比是衡量苜蓿品质的一个关键指标，而提高叶茎比则是苜蓿品质改良的重要手段。在本项目中，我们分离了蒺藜苜蓿PINNATE PENTAFOLIATA2（PPF2）的突变体株系。该突变体小叶数目由三个变为五个，表明PPF2基因抑制了复叶初级形态建成过程。PPF2属于BEL1-LIKE基因家族，可以与MtKNOXI家族成员在蛋白水平互作并抑制MtKNOX7的活性。另外，MtUFO和SGL1也参与了PPF2调控的复叶初级形态建成过程。本项目拟对PPF2与MtKNOXI、MtUFO/SGL1这两类复叶发育关键调控因子之间的分子机制开展研究，这将为建立苜蓿复叶发育基因调控网络模型奠定基础。同时我们拟将这些关键因子应用到紫花苜蓿的复叶模式分子设计中，最终为紫花苜蓿遗传改良提供新基因元件及新策略。

Class I KNOTTED-like homeobox（KNOXI）基因是控制叶片形态进化多样性调控网络的一部分。在单叶和复叶物种中，它们在叶片发育中的具有特定时空表达模式，并与叶片复杂性程度相关。然而，KNOXI基因并未参与某些豆科植物的复叶模式建立，其调控机制尚不清楚。在这里，我们发现了一种抑制MtKNOXI功能的新途径，该途径介导了蒺藜苜蓿（Medicago truncatula）从三出复叶模式到羽状五叶模式的转变。PINNATE PENTAFOLIATA2（PPF2）与MtKNOXI形成异源二聚体复合物，定位于细胞质中，阻止MtKNOXI进入细胞核直接激活下游靶基因UNUSUAL FLORAL ORGANS（MtUFO）的表达。同时，MtUFO的异位表达也会导致叶片呈现出羽状五叶模式。这些结果揭示了复叶植物叶片形态多样性的一个新的调控模式，即花发育的保守调控因子MtUFO和叶发育的保守调节因子MtKNOXI一同参与了羽状复叶模式的转变。