氢气通过NRAMP6维持离子稳态提高紫花苜蓿耐镉性的机理研究

Hydrogen gas (H2) is a novel gaseous signaling molecule found recently. Our results showed that cadmium could trigger endogenous H2 releasing in Medicago sativa seedlings. Moreover, exogenously applied hydrogen-rich water could not only mimic endogenous H2 releasing, but also alleviate cadmium toxicity in Medicago sativa (J Hazard Mater, 2013). Ion homeostasis was maintained by pretreatment of hydrogen-rich water. By using RNA-Seq approach, we further found that cadmium tolerance-associated ion transmembrane transport genes, such as metal cation transporter gene (NRAMP6) was regulated by hydrogen-rich water under cadmium exposure. In this project, an exploration in enzymatic resource of H2 production by cloning and prokaryotic expression analysis of Medicago sativa hydrogenase was carried out. Meawhile, Medicago sativa NRAMP6 gene was analyzed by eukaryotic recombinant expression, and its regulation mechanism by H2 as well as its role in maintaining ion homeostasis was also analyzed. Moreover, NRAMP6 over-expression or corresponding CRISPR/Cas mediated targeted mutagenesis plants were generated. By testing the cis-acting elements of NRAMP6 promoter region, combined with the responses of the loss- and gain-function plants on cellular ion homeostasis, cadmium transport, chelating and transport, the regulation mechanism of NRAMP6-mediated H2-enhanced cadmium tolerance were explored. Finally, the downstream key proteins as well as the signaling pathway leading to cadmium tolerance were analyzed through the combination of the phenotypic analysis and the proteomics approach. Together, above results will provide a new idea for the functional mechanism of the new signal molecule H2, and the cadmium tolerance mechanism as well as Medicago sativa production.

氢气（H2）是新发现的气体信号分子。我们研究发现，镉胁迫提高紫花苜蓿内源H2释放；富氢水不仅能模拟内源H2的释放，且能提高耐镉性（J Hazard Mater, 2013）；富氢水预处理下离子稳态得到重塑，进一步转录组测序发现，离子转运系统基因受到明显诱导，尤其是金属阳离子转运体基因（如NRAMP6）。本课题将克隆以及原核表达苜蓿氢酶基因，探查H2产生的酶学来源；进一步真核重组紫花苜蓿NRAMP6基因，分析其受氢气调控和维持细胞离子稳态缓解镉胁迫的机理；构建NRAMP6紫花苜蓿过表达及突变株系，结合NRAMP6启动子顺式作用元件分析以及细胞离子稳态、镉吸收、镉螯合和转运变化，发掘H2调控NRAMP6提高耐镉性的分子机理；通过对比上述材料根部蛋白质组，探查NRAMP6介导H2调控耐镉性的下游关键蛋白和信号通路。研究结果将为氢气作为信号分子的作用机制、耐镉性机理及苜蓿生产应用上提供依据。

氢气（H2）是新发现的具有生理活性的可能的气体信号分子。我们的研究发现，镉胁迫提高紫花苜蓿内源H2释放；富氢水不仅能模拟内源H2的释放，且能提高耐镉性；富氢水预处理下离子稳态得到重塑，进一步转录组测序发现，离子转运系统基因受到明显诱导，尤其是金属阳离子转运体基因（如NRAMP6）。本项目采用气相色谱测探查了不同浓度CdCl2处理下紫花苜蓿内源H2含量变化，结合恢复实验的表型，初步确认内源H2在镉耐性形成过程中的功能。通过对克隆到的MsNRAMP6基因的亚细胞定位分析发现其主要定位在质膜上，且其表达水平受到富氢水处理的调控。通过构建MsNRAMP6基因的拟南芥过表达株系，结合拟南芥同源基因Atnramp1突变体和野生型，分析了MsNRAMP6基因在介导氢气诱导的耐镉性中的作用，发现了氢气通过调控NRAMP6等离子转运体的水平，进而降低了镉胁迫下植物根部的镉积累，同时维持了镉胁迫下的离子稳态尤其是铁的稳态，并降低了镉引起的细胞氧化损伤。通过蛋白组及转录组分析挖掘了镉胁迫下氢气信号参与调控的关键细胞通路，并通过后续药理学实验和遗传学材料，验证了硫代谢是氢气诱导的耐镉性形成过程中的又一关键通路。此外，通过对紫花苜蓿MsNRAMP6启动子克隆及功能元件分析，发现一些可能的与氢气生物学效应相关的调控位点。相关结果从细胞离子稳态的维持和硫代谢通路相关的抗氧化和镉螯合及液泡区室化的角度解析了氢气介导耐镉性的相关机制。同时，项目课题组在多地和多家单位合作对富氢水的大田应用进行了实验，并获得便携式富氢水喷壶专利一项。研究结果将为氢气作为信号分子的作用机制、植物响应镉胁迫的机理以及苜蓿生产应用等提供依据。