家榆种子老化过程中ROS-类caspse-3途径的初步研究

以家榆种子为试材,在37℃、100%相对湿度下进行老化处理后,结合DAPI染色和细胞原位末端脱氧核苷酸转移酶标记法(TUNEL)、激光共聚焦技术以及生化分析,检测家榆种子人工诱导老化过程中细胞核、活性氧(ROS)和类半胱氨酸天冬氨酸蛋白酶3(caspase-3)活性的变化.结果显示:随着老化程度加深,种子细胞染色质皱缩、凝聚,继而解体并被排出体外;表皮中最先发现TUNEL凋亡核,而后逐渐延伸到子叶和胚轴;老化处理5d时种子活性氧信号最强,且其与程序性死亡相关事件的发生具有时空一致性,同时在胞浆中检测到较强的caspase-3活性.研究表明,家榆种子人工老化可导致细胞程序性死亡,且存在与ROS迸发及类caspase-3相关联的信号通路.

Programmed Cell Death of Ulmus pumila L.Seeds during Aging and ROS-caspse-3 like Pathway Mechanism

In this study,Ulmus pumila L.seeds were used to investigate characters of nuclear apoptosis,the reactive oxygen species and the activity of caspase-3 after artificially aging at 37℃ and 100% relative humidity(RH) by using confocal laser scanning microscopy(CLSM),DAPI(4′,6-diamidino-2-phenylindole) and TUNEL staining method and caspase-3 detection kit,respectively.The results indicated that the nuclear chromatin appeared to be condensation and shrinkage with the increasing of the ageing time.Some nuclei dissociated into pieces and were released from the cells.The apoptosis was firstly observed in the epidermal cells,and was later found in the cotyledons and hypocotyls.This research also showed the distinct spatial-temporal signature of ROS during ageing coincided with the changes in PCD(programmed cell death) hallmark features,and the signal intensity of Reactive oxygen species(ROS) was gradually increased at 5 d.The activity of caspase was higher in the cytoplasm.Our data indicate that the ROS and caspase-3-like also act as PCD mediators in the process induced by aging.