细胞程序性死亡在半夏干旱倒苗中的作用及调控机制研究

Drought is one of the most important environmental restrictive factors that cause sprout tumble of pinellia ternata. Development of pinellia ternata production is restricted due to the lack of the understanding of molecular mechanism of sprout tumble caused by drought. In our preliminary study, typical hallmarks of programmed cell death (PCD) such as nucleus condensed chromatin and caspase-3 activities increased were found during the process of pinellia ternata sprout tumble caused by drought. PCD may play major role in the process of sprout tumble regulation. We will conduct further study with simulated drought treatment by polyethylene glycol (PEG). DNA ladder and DAPI dying will be detected to confirm the characteristic spatio-temporal expression of PCD during the process of pinellia ternata sprout tumble. The effect of PCD in this process will be certified by comparing drought tolerance with occurrence of PCD among the different pinellia ternata population. Through pretreatment with signal factors like exogenous Ca2+ and inhibitors which could block signal transduction pathway the changes of mitochondria physiological biochemical and PCD will be detected. As a result, this study will illustrate the relationships from drought stress—signal factors—signal transduction pathway—mitochondria physiological to PCD and confirm the regulatory mechanism of PCD in pinellia ternata sprout tumble under drought stress.In summary, this study will expound the molecular mechanism of pinellia ternata sprout tumble caused by drought so as to provide a theoretical basis of how to prevent this phenomenon.

干旱是引起半夏倒苗造成其减产的重要环境限制性因子。目前，由于对半夏干旱倒苗分子机理了解的欠缺，严重制约了半夏生产的发展。申请者前期研究发现，半夏干旱倒苗过程中出现细胞核染色质凝集、Caspase-3活性升高等典型细胞程序性死亡（PCD）特征，PCD可能参与该过程的调控并在其中起关键作用。本项目拟以PEG模拟干旱处理，通过DNA 梯、DAPI染色等方法确定PCD在半夏干旱倒苗中的时空表现特征，比较不同居群半夏耐旱性与PCD发生的关系，明确PCD在其中的作用；通过组合添加Ca2+等外源信号分子供体及MAPK等信号转导途径相关酶抑制剂，检测分析干旱胁迫、信号因子、信号转导途径、线粒体生理生化变化与PCD之间的联系，从而明确PCD在半夏干旱倒苗中的调控机制。通过该研究，可揭示半夏干旱胁迫和倒苗的分子机理，为防止半夏干旱倒苗提供重要的理论依据。

干旱是引起半夏倒苗造成其减产的重要环境限制性因子。目前，由于对半夏干旱倒苗分子机理了解的欠缺，严重制约了半夏生产的发展。申请者前期研究发现，半夏干旱倒苗过程中出现细胞核染色质凝集、Caspase-3活性升高等典型细胞程序性死亡（PCD）特征，PCD可能参与该过程的调控并在其中起关键作用。为了解半夏干旱倒苗的机理，以30% PEG 6000模拟干旱胁迫，检测了不同处理时间下半夏细胞的生理和形态变化。结果表明：随着干旱胁迫时间的延长，半夏茎的叶绿素含量及水势不断下降，相对电导率、可溶性糖和MDA的含量则不断升高。干旱处理4 d，半夏开始倒苗，至8 d时大幅度倒苗，倒苗程度约为73%左右。干旱处理2 d，半夏细胞核核染色质开始凝集，核固缩边缘化，呈新月状等不规则形状，表现出明显的细胞程序性死亡特征。腺苷酸转移酶（ANT）基因表达逐渐受到干旱的抑制，随着Caspase-3酶活性急剧升高，细胞死亡率不断增加。以干旱胁迫下半夏发生“倒苗”时的茎段作为研究材料，采用 Illumina Hiseq 2500 平台进行转录组双向测序，并对数据进行生物信息学相关分析，最终筛选得到差异表达基因 4 052 个，其中 2080 个 DEGs 表达上调，1 972 个 DEGs 表达下调。KEGG 通路富集分析显示，差异表达基因主要集中在核糖体、碳代谢、淀粉和蔗糖代谢、植物激素信号转导和氨基酸生物合成等过程；基因表达水平分析显示，半夏中编码植物激素代谢与信号转导、氧化还原相关酶、热激蛋白和液泡加工酶等基因在干旱胁迫处理后表达水平显著上升，多数编码水通道蛋 白的 DEGs 表达水平下降。由此可见，半夏干旱倒苗的本质是细胞发生程序性死亡，这可能是一种半夏抵御不良环境的自我休眠保护机制。