自然和人工老化大豆种子胚轴细胞的时空特异性死亡研究

Seed aging is accompanied with cell death. However, there were only very few published works concerning cell death taking place during seed aging. Among those few researches, seeds were artifically aged under relatively high temperature and relative humidity (RH) instead of natural aging at room temperature or lower temperature, thus comparisons between naturally and artifically aged seeds are needed. Moreover, in most researches whole seed/embryo were used as materials, while differences among tissues/organs were neglected. Our previous work with aged soybean seeds showed that the cells from different organs of the axis proformed differently during seed aging, with cells from the radicel died at first. We also found that the mechanism of seed aging might differ with seed aging conditoin. In this project we will compare the tmporal- and spatial-specific cell death of soybean seeds being naturally and artifically aged. TTC and Evans blue dyeing techniqe are used to assess the viability of cells from different parts of the axis, and ultimately make it clear that which part of the axis die first upon seed aging. Using a fluorescence dyeing kit, we can tell whether the cells die by way of programmed cell death (PCD) or necrosis. The proteome of cells that temporally and spatially died during seed aging will be compared to get a better understanding of the seed aging mechanism. We believe that a better understanding of seed aging will bring big progress in seed storage security.

种子老化过程伴随着细胞死亡，然而目前对细胞死亡事件研究较少，且缺乏对种子各部位细胞死亡的时空差异的研究，亦较少关注对自然和人工老化的对比研究。我们前期工作发现，大豆种子人工老化过程中胚根细胞比其他部位细胞更早死亡，并发现自然和人工老化机制可能不同。本项目以此为切入点，以自然和人工老化的大豆种子为试材，利用活力染色准确定位老化胚轴最先死亡的细胞，采用细胞凋亡/坏死荧光染色方法检测细胞死亡方式，通过DNA琼脂糖凝胶电泳和非损伤微测技术测定K+和Ca2+离子变化以验证细胞死亡方式，并应用蛋白质组技术对比其蛋白差异，阐明种子老化过程中细胞时空特异性死亡特征及调控机理，以进一步揭示种子老化机理，进而为我国作物种子安全贮藏提供理论与实践指导。

本项目研究目标是通过染色手段确定大豆种子老化过程中细胞死亡的时空变化，明确决定种子存活与否的关键细胞及其在种子老化过程中的死亡方式，分离并鉴定与胚轴细胞死亡时空特性相关的特异蛋白。研究过程中，我们分别采用TTC和Evans Blue染色法，对不同老化程度的大豆胚轴进行染色，确定了种子老化过程中，胚轴死亡从胚根扩展至下胚轴。证明了老化胚轴中细胞死亡存在时空差异。采用PI荧光染色，验证了细胞死亡的时空差异；采用TUNEL荧光染色、DNA laddering、 K+和Ca2+等检测，发现死亡细胞中同时存在PCD和坏死，PCD主要发生于较低活力胚根部位，且自然老化和人工老化两种条件下细胞死亡模式存在差异。利用双向电泳和质谱鉴定技术，分析老化过程中非同步死亡细胞的蛋白质组差异，发现种子老化过程中，胚根和下胚轴两部分的表达蛋白存在差异，胚根中表达量改变的蛋白点数量更多，且蛋白质功能分类也不同，表明胚根和下胚轴基因表达存在明显差异。为验证该结果，我们增加了转录组实验，结果证明老化过程中胚根和下胚轴中有大量的基因、蛋白差异表达，其中脂质、微管和细胞骨架相关的基因最为明显。对照项目目标，基本完成各项研究任务，发表SCI论文1篇，另有1篇SCI论文正在撰写中。培养研究生1名。