基于细胞凋亡抑制途径的酵母耐铝性及其胞内钙信号调控分子机理研究

项目名称： 基于细胞凋亡抑制途径的酵母耐铝性及其胞内钙信号调控分子机理研究

项目编号： No.30870034

项目类型： 面上项目

立项/批准年度： 2009

项目学科： 轻工业、手工业

项目作者： 朱睦元

作者单位： 浙江大学

项目金额： 30万元

中文摘要： 铝(Al)毒是酸性土壤中限制作物生长和产量的重要因素之一，本研究以酿酒酵母为材料，研究胞质Ca2+信号在Al3+诱导的PCD中的调控作用及其抗Al毒的内部耐受机制。我们发现液泡Ca2+泵Ca2+-ATPase PMC1的突变体(pmc1)对Al毒的敏感性高于野生型，Al3+处理条件下 pmc1发生凋亡的细胞数目显著多于野生型。过量表达PMC1或者将凋亡抑制基因(Bcl-2、Ced-9或PpBI-1)转入野生型和pmc1突变体，能降低胞质Ca2+水平，增强对Ca和Al胁迫耐受性。胞内Ca2+螯合剂BAPTA-AM预处理能抑制Al3+诱导胞质Ca2+的上升，降低Al对细胞的毒害作用。用荧光定量RT-PCR检测Ca信号途径因子的表达情况，发现Al3+处理时calmodulin (CaM) 和phospholipase C (PLC)的表达水平下调，而在Ced-9过表达株系及BAPTA-AM预处理时，这种下调作用被抵消。总之，Al诱导的胞内Ca2+信号的负调控是一种新的Al内部耐受机制，可以为其他生物耐铝性改良提供新思路和新方法。

中文关键词： 酵母；铝；钙信号;细胞凋亡

英文摘要： Aluminum (Al) toxicity is a main factor to limit crops growth and products under acid soil. In this study, we found that a yeast mutant, pmc1, lacking the vacuolar calcium ion (Ca2+) pump Ca2+-ATPase (Pmc1p), was more sensitive to Al treatment than the wild-type strain. Overexpression of either PMC1 or an anti-apoptotic factor, such as Bcl-2, Ced-9 or PpBI-1, decreased cytoplasmic Ca2+ levels and rescued yeast from Al sensitivity in both the wild-type and pmc1 mutant. Moreover, pretreatment with the Ca2+ chelator BAPTA-AM sustained cytoplasmic Ca2+ at low levels in the presence of Al, effectively making the cells more tolerant to Al exposure. Quantitative RT-PCR revealed that the expression of calmodulin (CaM) and phospholipase C (PLC), which are in the Ca2+ signaling pathway, was down-regulated under Al stress. This effect was largely counteracted when cells overexpressed anti-apoptotic Ced-9 or were pretreated with BAPTA-AM. Taken together, our results suggest that the negative regulation of Al-induced cytoplasmic Ca signaling is a novel mechanism underlying internal resistance to Al toxicity, which can provide a new approach to improve crop tolerance under Al stress.

英文关键词： Yeast; Aluminum; Calcium signal; Apoptosis