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

项目名称： 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

项目编号： No.31501237

项目类型： 青年科学基金项目

立项/批准年度： 2016

项目学科： 食品科学、农学基础与作物学

项目作者： 崔为体

作者单位： 南京农业大学

项目金额： 20万元

中文摘要： 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。

中文关键词： 其它作物；重金属；耐性；信号；组学

英文摘要： 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.

英文关键词： Medicago sativa;Heavy metal;Tolerance;Signal;Omics