被子植物胚柄细胞程序性死亡的分子机理

The suspensor is a terminally differentiated and transcient structure that connects the embryo to surrounding tissues, and severs as a conduit for nutrients and growth regulators for embryo proper development, and disappeared as seeds matured. The process of suspensor disappearance in angiosperms has been believed to go through programmed cell death program, and markers of eukaryotic programmed cell death, such as DNA fragmentation, nuclear degradation, and caspase-like activities, have been observed in some plants. However, the key components regulating suspensor degeneration, the process and mechanisms responsible for converting the suspensor from a differentiation program to a cell death program are poorly understood in angiosperms. In provious experiment we isolated apical and basal cells of tobacco by using laser-controlled micro-dissection, and constructed cell type-specific cDNA libraries. We characterize a gene from basal cell cDNA library designed as NtPCD1 that accumulates specifically within the suspensor from two-celled proembryo to eight-celled embryo stage. The NtPCD1 paly a important role in regulating programmed suspensor cell death of suspensor. Down-regulation expression of NtPCD1 paly a important role in regulating programmed suspensor cell death of suspensor. Down-regulation expression of NtPCD1 result in suspensor death in advance. We would investigate the molecular mechanism of programmed suspensor cell death by studing the function of NtPCD1.

胚柄是被子植物胚胎发生过程中出现的一个短命结构，有支撑、固定胚胎在胚囊中的位置﹑向胚体运输营养物质和激素等作用，对早期胚胎发生中细胞命运决定，胚胎顶基轴向确定至关重要。胚柄细胞发育最显著的特点是其适时地解体死亡。前人研究结果表明胚柄细胞细胞的降解可能是一个程序性死亡的过程。在胚柄细胞降解的过程出现染色体浓缩﹑DNA核小体的断裂﹑细胞核固缩及核仁变小等类似于动物细胞凋亡的特征。然而，到目前为止，胚柄细胞程序性死亡的分子证据依旧缺失，对其促发因子和调控的分子机制更不清楚。本研究工作基于本实验室烟草顶﹑基细胞文库构建和测序﹑RT-PCR筛选特异表达基因等前期基础，克隆了胚柄细胞特异表达的NtPCD1基因，该基因对胚柄细胞程序性死亡促发时间的调控起着关键的作用。NtPCD1表达水平的降低导致胚柄细胞提前降解。我们将以此为切入点，系统研究胚柄细胞程序性死亡的分子机理。

本课题的研究目标是：通过对NtPCD1的功能分析，明晰胚柄细胞程序性死亡的分子机制。在受基金委资助的四年中，我们认真地按照原计划内容进行研究，并达到了预期目的。我们通过对胚柄、胚体差异表达基因的分析发现了一个仅在胚柄泡状细胞特异表达的基因NtPCD1，该基因的下调导致胚柄提前发生程序性死亡，而其过表达会延迟胚柄的程序性死亡。这表明NtPCD1是胚柄程序性死亡的关键控制基因。生物信息学分析表明NtPCD1编码半胱氨酸蛋白酶抑制剂，其下游的底物可能为半胱氨酸蛋白酶。我们系统研究了NtPCD1基因的生化特性与生物学功能。通过体外生化试验，体内验证等手段发现了与NtPCD1互作的半胱氨酸蛋白酶NtCP14。与NtPCD1的功能相反，NtCP14基因的下调延迟胚柄的程序性死亡，而其过表达会导致胚柄提前发生程序性死亡。这些结果表明NTPCD1-NtCP14两个基因的拮抗行使分子开关作用，决定胚柄细胞的发育与死亡。在该项目资助下，我们在Plos Biology，DNA Research等SCI刊物上发表论文五篇。