干旱胁迫下八棱海棠PCD特征检测及类caspase基因片段的克隆与分析

Detection of programmed cell death and cloning of caspase-like gene fragment from Malus robusta under drought stress

【目的】为了探明类caspase蛋白酶参与植物PCD的机制,【方法】以八棱海棠(Malus robusta Rehd.)实生苗为试验材料,对植株进行轻度干旱胁迫,检测叶片PCD的发生,并应用RT-PCR技术克隆类caspase基因。【结果】结果表明,轻度干旱胁迫后3周的叶片出现细胞染色质凝聚、胞质皱缩、细胞核变形等细胞凋亡的形态学特征。提取叶片DNA,观察到DNA呈现明显的"DNA Ladder",表现出典型的细胞程序性死亡的生化特征。在此基础上提取叶片RNA,采用RT-PCR获得长度为555 bp的类caspase基因片段。【结论】经同源性分析该片段与其他果树的同源序列相似性超过80%,提示该区段为保守序列。比对苹果基因组,八棱海棠基因组中存在半胱氨酸蛋白酶基因,该基因为单拷贝,并且存在2个同源基因,这为揭示干旱胁迫下八棱海棠调控PCD的机理及环境适应的分子机制奠定了基础。

[Objective] The objective of the study is to elucidate the mechanism of caspase-like proteases involved in the plant PCD. [Method] Under drought stress, the features of programmed cell death （PCD） were detected in the treated Malus robusta Rehd. leaves, and the caspase-like gene was cloned by RT- PCR. [Result] The results showed that the features of programmed cell death （PCD） were found in the treated Malus robusta Rehd. leaves, such as chromatin condensation, cytoplasmic shrinkage, karyon distorted. One characteristic of PCD in animals and plants is the cleavage of nucleosomal DNA into fragments of about 180-200 bp which is usually observed in the form of a ＇DNA ladder＇ during PCD. The pattern of DNA cleavage extracted from the treated leaf was analyzed using gel electrophoresis. The results showed the typical characteristic ＇DNA ladder＇. Then a 555 bp fragment of caspase-like gene was isolated by RT-PCR using RNA extracted from the treated leaf as template. [Conclusion] The similarity of the frag- ment of caspase-like geneamong Malus robusta Rehd. and other fruit tree reached over 80%, which suggested that this section was conservative sequence. Reference to the apple genome, it indicated that the caspase-like gene probably has only one single copy and two homologous genes in the genome of Malus domestica. These results laid the foundation for the reveal of the PCD mechanism and molecular mechanism of adaptation to environment of Malus robusta Rehd. under drought stress.