自噬在小麦抵抗镉胁迫诱导的凋亡中的作用及机制研究

Autophagy is one of the stress-tolerance mechanisms in plant. The activation of autophagy strictly depends on the temporal and spatial expression of autophagy related genes (ATG). As the core element of autophagy activation, ATG6 gene has been the effect target in bothh function research of autophagy and playing an important role in apoptosis regulation. By our prophase research，we have found both autophagy and apoptosis were activated by Cd (cadmium, Cd) stress. ATG6 gene silenced-wheat showed autophagy deficiency and sensitive to Cd stress. While the ATG6 gene over-expressed-wheat showd tolerance to Cd stress. The expression of ATG6 gene could effectively mitigate toxicity of Cd to wheat plant, and resulted in an increased percentage of apoptosis in contrast to other Cd-treated groups. Based on the previous results and technical platform of our laboratory, we choose wild type, ATG6 gene silenced and over-expressed wheat plant as experimental materials for experiment. We will investigate the mechanism research of wheat ATG6 gene in mediating the response of wheat (Triticum aestivum L.) to Cd stress. Firstly, we will investigate the expression of ATG6 gene and the subcellular localization of ATG6 protein responsed to Cd Stress in wheat. Secondly, we will study the influence of ATG6 on Cd2+ uptake, metabolism and accumulation in wheat. Thirdly, we will investigate the relationship between ATG6 gene expression and apoptosis in wheat responded to Cd stress. Finally, we will study the mechanisms of ATG6 gene expression regulationg Cd -induced apoptotic signalling pathways. In the course of the investigation, some methods and techniques including non-invasive micro-test technique (NMT), inductively-coupled plasma emission spectrometer (ICP-AES), virus induced gene silence (VIGS), immunofluorescence localization techniques and western blotting techniques, ect. will be used. With the uncovering of the molecular basis of ATG6 gene in resistance to cadmium-induced apoptosis of wheat, the analysis of the test results will provide a new insight into understanding of the effects and mechanisms of autophagy in resistance to cadmium-induced apoptosis of wheat.

自噬是植物进化中形成的耐逆境胁迫的机制之一。自噬激活严格受自噬相关基因（ATG）调控，ATG6基因是激活自噬的核心元件，同时调控凋亡。前期研究表明镉（Cd）诱导小麦发生自噬和凋亡；ATG6基因沉默植株呈典型自噬缺陷，根尖细胞凋亡率高，对Cd耐受性低；过表达ATG6基因植株根尖细胞凋亡率下降，对Cd耐受性增强。据此，本项目以小麦野生型、ATG6基因沉默和过表达植株为材料，开展如下研究：（1）Cd胁迫下ATG6基因的表达模式及其表达蛋白的亚细胞定位；（2）ATG6基因表达对Cd2+吸收、转运、累积的影响；（3）ATG6基因表达与凋亡的关系；（4）ATG6基因调控Cd诱导小麦凋亡的信号途径。本项目采用非损伤微测、元素分析、病毒诱导的基因沉默、荧光染色、western blotting等方法，通过阐明ATG6基因调控小麦抵抗Cd胁迫的机理，揭示自噬在小麦抵抗Cd胁迫诱导的凋亡中的作用及机制。

自噬是植物进化中形成的耐逆境胁迫的机制之一。自噬激活严格受自噬相关基因（ATG）调控，ATG6基因是激活自噬的核心元件，同时调控凋亡。本项目以重要农作物小麦为研究对象，以小麦对镉胁迫环境的响应为研究内容，研究小麦幼苗对镉的吸收与积累的规律，研究Cd诱导的小麦根尖细胞凋亡，Cd胁迫激活小麦根细胞自噬，及自噬正调控小麦根尖耐镉胁迫的过程等4方面的内容。结果表明，不同小麦品种对镉耐性有差异，Cd 胁迫诱导小麦根尖发生细胞凋亡和细胞自噬，采用自噬特异性抑制剂3-MA，探讨自噬对小麦细胞凋亡的影响，采用 PCD抑制剂 Ac-DEVD-CHO ( ADC)作用小麦根后，LTD 荧光染色法检测细胞自噬水平的变化。发现加入3-MA 和ATG6沉默抑制小麦细胞的自噬发生后，细胞的凋亡水平明显上升；加入 ADC 作用小麦后，小麦根吸收 Cd 增多，但转移到叶中的量减少，缓解了对叶片的毒害作用。这些结果表明，自噬能够负调控 PCD，小麦响应 Cd 胁迫早期凋亡的发生有利于限制 Cd 向地上部分转移，可能是小麦耐 Cd 胁迫的一种机制。研究结果初步了揭示自噬在小麦抵抗Cd胁迫诱导的凋亡中的作用及机制。