BR信号调控拟南芥幼年向成年阶段转变发育的研究

The juvenile-to-adult phase transition (J-to-A transition) is a critical process in plant development, and it is regulated by a highly conserved miR156-SPLs-miR172 pathway. However, how upstream factors regulate this conserved pathway remains little known. We will carry out this study using a del2 (Delayed juvenile to adult transition mutant 2, del2) mutant isolated from an EMS-mutagenized mutant screen. The del2 mutant exhibited some typical characteristics of juvenile traits including more juvenile and rounder leaves. Preliminary study revealed that del2 is allelic to dwf5 defective in Brassinosteroid (BR) biosynthesis. Gene expression analysis showed that the primary transcript of miR172, MIR172B, was significantly downregulated in del2, suggesting that BR is involved in the miR156-SPLs-miR172 pathway. In this study, we will employ genetics and molecular biology to study the function of BR in the regulation of the miR156-SPLs-miR172 pathway. We try to elucidate how BR converge to the miR156-SPLs-miR172 pathway to regulate plant vegetative development in order for us to explore more regulatory factors upstream of this conserved pathway.

植物幼年向成年阶段转变是一个重要的发育过程，该过程受保守的miR156-SPLs-miR172途径所调控，但该途径上游调控网络研究较少。本项目以EMS诱变筛选获得的拟南芥del2（Delayed juvenile to adult transition mutant 2, del2）突变体为材料，del2突变体表现为幼年叶数目增多、叶片变圆、幼年期延长，为典型幼年向成年阶段转变延迟突变体。前期工作确定del2为油菜素内酯 （Brassinosteroid, BR）合成缺陷突变体，为dwf5等位突变。基因检测发现del2中miR172前体MIR172B显著下调，确定BR参与调控miR156-SPLs-miR172途径。本项目将利用遗传学和分子生物学来研究BR信号调控miR156-SPLs-miR172途径的作用节点和作用机制，揭示BR调控植物营养期发育的新功能，并拓展该途径的上游调控网络。

成年发育（即幼年向成年阶段转变过程）是植物营养期的关键发育过程，该过程与作物株型、抗逆性等关键经济性状密切相关；此外，加速作物成年发育，对于缩短育种周期也至关重要。植物成年发育受保守的miR156-SPLs-miR172途径所调控，外界环境条件（如生物和非生物胁迫、弱光条件）和内源因子（如糖、GA、JA）等均可通过该途径影响植物成年发育，但对于miR156-SPLs-miR172途径的调控网络仍旧知之甚少。.本研究基于一份拟南芥EMS诱变获得的成年延迟突变体del2，主要研究内容和结果如下：①图位克隆和转基因回复实验，确定del2为油菜素内酯（Brassinosteroid，BR）合成缺陷突变体，为dwf5等位突变。②表型统计显示del2具有叶圆、叶下表皮毛发生延迟，为典型成年发育延迟的缺陷突变体，证明BR信号可正调控拟南芥成年发育。③基因检测显示del2中MIR172B显著下调，双突变体遗传分析确定del2的成年发育延迟源于MIR172B表达量降低。④瞬时诱导实验证明，del2背景下，GR-rSPL9诱导MIR172B缺陷。⑤依据BR信号途径作用模式，结合del2上述实验结果，酵母互作分析确定BIN2可互作SPL9，且负调控SPL9蛋白稳定性；rSPL9 del2双突变体表型统计分析，验证上述生化结果。⑥BR/GA互作调控成年发育，发现BR信号可影响GA20ox1和GA3ox1，进而通过GA-DELLA-SPL9途径调控拟南芥成年发育。.本研究证明BR激素可调控拟南芥成年发育过程，作用节点为SPL9-MIR172B，BR信号途径关键负调控因子BIN2可互作SPL9，通过磷酸化修饰SPL9并降低其稳定性，进而延迟拟南芥成年发育。本研究丰富了植物成年发育的基因调控网络，特别是激素信号调控成年发育的具体作用机制，对于理解和人为调控植物成年发育过程具有参考意义。