拟南芥AT hook家族基因（AHL5/9/11/12）维持茎顶端分生组织功能的分子机制

Shoot apical meristem (SAM) is the main source of stem cells where all aerial organs are produced. In order to fulfill this function, the meristem must maintain the balance between the self-renewal of a reservoir of central stem cells and organ initiation from peripheral cells. Although some important genes have been reported to regulate the development of SAM, the underlying mechanism has not been fully elucidated. The AT-hook family proteins with a small DNA-binding motif are implicated in the transcriptional co-regulation of target genes via regulating the chromatin architecture, which play important roles in plant growth and development. Transgenic knockdown of AT hook genes AHL5/9/11/12 in Arabidopsis leads to loss of apical dominance, retarded growth, and altered leaf shape, which are similar with loss-of-function mutants of the key regulators of SAM, SHOOT MERISTEMLESS (STM) and WUSCHEL (WUS), suggesting that the four AT-hook genes may play pivotal roles in the regulation of plant stem cell. Based on this, the biological functions of the four AT-hook family genes will be systemically investigated with CRISPR-Cas9 technology producing mutants, molecular genetic, biochemical, and transcriptomic approaches, which will help us better to reveal the function of AHL5/9/11/12 in SAM. The study will be a substantial aid in understanding the mechanism of plant stem cell maintance.

茎顶端分生组织是植物地上部组织和器官发生的根源，分生组织细胞分裂和分化之间平衡的维持依赖于复杂的基因调控网络和信号传导途径。虽然调控茎顶端分生组织发育的一些重要基因已经报道，但对于多基因网络调控其发育的机制了解较少。AT hook是一种小型的DNA结合蛋白基序，该家族蛋白在植物的生长和发育过程中发挥重要作用。申请人前期研究发现，AT hook (AHL5/9/11/12)转基因植株表现出顶端优势缺失、生长缓慢和叶形改变等表型，与干细胞维持关键基因STM和WUS的突变表型相似。本申请项目拟在此基础上，通过CRISPR-Cas9等多基因敲除技术获得相应突变体，综合运用分子遗传学、细胞生物学、生物化学、转录组学等方法，深入研究AHL5/9/11/12与已有干细胞维持关键因子STM和WUS之间关系，揭示AT hook基因家族在茎顶端分生组织中的作用，进一步丰富植物干细胞维持与调控机制。

植物叶片衰老是叶片发育的最后一个过程，受到很多内在因子和外界因素影响。虽然植物叶片衰老机制研究取得了一定进展，但更多调控因子的挖掘仍是目前研究的重要科学问题和热点。AT hook是一种小型的保守DNA结合蛋白基序，该家族蛋白在植物的生长和发育过程中发挥重要作用。本项目研究发现拟南芥AT hook家族成员AHL9和AHL11超表达时导致叶片早衰，叶绿素含量明显减少。同时在水稻中异源超表达AHL9和AHL11也表现出叶片早衰的表型。AHL9和AHL11启动子启动GUS和RT-qPCR表达模式结果显示幼龄叶片的表达量比成熟和衰老叶片表达量更强。体外处理数据显示，AHL9和AHL11超表达拟南芥在ACC和黑暗处理后叶片变黄更快，叶绿素减少更多，而ABA处理则无明显影响。另外，拟南芥乙烯合成或信号转导相关基因（AP2、ERF39、ACO2）及叶片衰老相关基因表达（NAC100、NAC79、LOX1等）在AHL9和AHL11超表达转基因植株中发生了明显改变。综上所述，我们的研究表明AHL9和AHL11作为两个重要因子调控叶片衰老过程。该工作将为作物成熟和衰老等重要农艺性状的改良和分子育种提供重要的理论依据和基因资源。