拟南芥MAN3介导的镉积累和耐受的分子机制研究

Previously, we cloned Arabidopsis MAN3 gene involved in the regulation of cadmium accumulation and tolerance, which encodes an 1,4-β-mannanase. We found that MAN3 regulates the glutathione (GSH)-dependent phytochelatin (PC) synthesis pathway by coordinated activation of expression of genes involved in PC biosynthesis, which is triggered by mannose-mediated signaling, and thereby enhances Cd accumulation and tolerance in Arabidopsis. However, little is known about signaling components function in upstream and downstream of MAN3 in response to Cd stress. Recently, we found that loss function of MBL1 led to Cd sensitivity, and thereby speculated that MAN3 might trigger expression of genes involved in PC biosynthesis by interaction between mannose and MBL1 to regulate the response of plant cells to Cd stress. In addition, we have also screened a MYB transcription factor (designated as TFx) that possibly directly regulates in transcription of MAN3 gene, and found that the tfx mutants showed enhanced Cd sensitivity. In this project, we proposed to identify and analyze upstream TFx and downstream MBL1 of MAN3 in response of plants to Cd stress, and thereby uncover a TFx-MAN3-mannose-MBL1 signaling pathway and its action mechanism involved in the regulation of the response of plants to Cd stress.

前期克隆了一个编码拟南芥1,4-β-甘露聚糖内水解酶的基因MAN3，发现其通过甘露糖（mannose）介导的信号来调控谷胱甘肽（GSH）依赖的植物螯合肽（PC）合成途径上相关基因表达，从而增加植株对镉（Cd）的积累和耐受。然而， MAN3上游和下游的信号转导组分尚不清楚。最近我们发现一个mannose特异结合蛋白MBL1功能缺失突变体对Cd胁迫敏感，据此推测MAN3可能通过mannose与MBL1互作来启动GSH依赖的PC合成途径上相关基因表达，从而实现植物细胞对Cd信号的应答反应；此外，还筛选了一个可能直接调控MAN3基因转录的MYB转录因子（暂命名为TFx），该基因敲除的突变体tfx对Cd胁迫敏感。本项目拟研究植物响应Cd胁迫过程中MAN3的上游TFx和下游MBL1，阐明一条植物响应Cd胁迫的信号转导通路TFx-MAN3-mannose-MBL1，揭示其调控植物对Cd胁迫响应的机制。

前期研究表明，由MAN3介导的甘露糖在植物应对镉胁迫响应中扮演着重要的角色。然而，所涉及的潜在分子机制和信号传导途径尚不清楚。在本研究中，我们发现拟南芥MYB4-MAN3-Mannose-MNB1信号通路参与植物镉积累和耐受的调控。研究表明，MNB1功能缺失突变体降低了植株对Cd的积累和耐受性，而MNB1的过表达则显著增强了植株对Cd的积累和耐受。与之一致地是，无论在有无镉胁迫条件下，mnb1突变体中GSH依赖的PC合成途径相关基因如GSH1、GSH2、PCS1和PCS2的表达显著下降，而在MNB1过表达植株中得到相反的结果。这表明MNB1基因是通过GSH依赖的PC合成途径来正向调控植株对镉的积累和耐受。此外，我们发现甘露糖能够与MNB1特异性结合并且该结合依赖于MAN3介导的镉耐受。进一步分析表明，MYB4能直接激活MAN3的转录，并且MYB4基因也是通过GSH依赖的PC合成途径来正向调控植株对镉的积累和耐受。遗传分析发现，MAN3的过量表达能恢复myb4突变体对镉敏感的表型，但不能恢复mnb1突变体对镉敏感表型，然而MNB1的过量表达则能恢复myb4突变体的镉敏感表型，这些结果说明MAN3作用于MYB4下游，但作用于MNB1上游。总之，我们的研究结果证实了在拟南芥中MYB4-MAN3-Mannose-MNB1这一信号通路通过GSH依赖的PC合成途径来调控镉的积累和耐受。该研究不仅揭示了植物对重金属胁迫响应调控的新机制，而且为植物修复基因工程提供新的基因资源和技术途径。已在国际学术期刊上发表基金标注的SCI论文8篇，申请发明专利6项，相关成果获奖2项，培养研究生16人，待发表SCI论文2－3篇。