三七根硝酸还原酶磷酸化及与14-3-3蛋白互作参与NO介导Cd积累的调控机理研究

Panax notoginseng (Burk.) F.H.Chen is one of the precious traditional herbs in China. The roots are used for the treatment of cardiovascular disease, inflammation, trauma and hemorrhage. However, the high capacity of its roots to accumulate cadmium (Cd) poses a potential risk to human health. Cd contamination of P. notoginseng roots is directly correlated with its medicinal safety, making this a primary obstacle for its international trade. Thereby, minimizing or preventing Cd contamination of P. notoginseng roots is an urgent problem that remains to be solved. The preliminary study for this project showed that nitrate reductase (NR)-dependent NO biosynthesis enhanced Cd accumulation in Panax notoginseng root. Moreover, the interaction between the 14-3-3 protein and NR is involved in this process by negative regulation of the NR activity and NO production. On the basis of these results, the phosphorylation and interaction of NR with the 14-3-3 protein in the regulation of NR activity and modulation of NO production will be further determined in Panax notoginseng root in this project. Furthermore, the mechanism of the NR-dependent NO production in the enhancement of Cd accumulation will be clarified by determining the subcellular contents of Cd and the cell wall (CW) components (pectin and hemicellulose) in Panax notoginseng root. Finally, the regulatory mechanism will be further verified and clarified by overexpression or RNAi-mediated downregulation of the 14-3-3 protein-encoding gene in Panax notoginseng suspension cells. The implementations for the project will not only be helpful for us to better understand the physiological and molecular mechanisms of Cd-induced NO production in plant, but also provide a new research insight and a novel strategy to prevent Panax notoginseng from Cd contamination through biotechnological approaches.

三七是我国传统名贵中药材，主要以根部入药。三七根有较强的镉（Cd）累积能力，根中Cd含量偏高已成为制约我国三七出口的主要因素之一。因此，如何控制三七Cd污染是当今三七药品安全和国际贸易面临的首要问题。针对这个问题，我们前期研究发现依赖于硝酸还原酶途径产生的NO促进三七根中Cd的累积，且14-3-3蛋白和硝酸还原酶的相互作用参与这个调控过程。本项目在此研究基础上，拟通过考察Cd处理下三七根中硝酸还原酶的磷酸化水平及其与14-3-3蛋白互作对该酶活性和NO含量的影响，同时检测由此途径调控产生的NO对Cd的亚细胞水平含量和细胞壁组份的影响，揭示硝酸还原酶的磷酸化及其与14-3-3蛋白互作参与NO促进三七根中Cd积累的调控机制；该机制将在转基因三七细胞中得到验证。本研究不仅可以使我们深入理解Cd诱导植物产生NO的生理及分子机理，也为今后通过生物技术手段控制三七Cd污染提供新的研究思路和策略。

镉（Cd）是环境中生物毒性最强的重金属污染物之一，Cd污染不仅限制作物生长和产量，大量累积于作物中也对人体健康造成潜在威胁。三七是云南省特色中药材，重金属超标是三七药品安全和社会关注的焦点。本项目分析了Cd在三七根中累积的调控机理，为控制三七Cd污染的绿色可持续发展提供新策略。.本项目的具体研究结果如下：（1）Cd通过抑制14-3-3蛋白表达及其与NR互作，降低NR的磷酸化水平，提高NR的活性而增强NO的产生；然而Mg可以有效的增强Cd胁迫下14-3-3表达及其与NR互作水平，降低NR活性和与之相关的NO产生；（2）Cd胁迫下外源添加NO供体SNP显著提高Cd在三七根中的累积，然而外源添加NR抑制剂tungstate和Mg降低了Cd诱导的三七根中NO的累积和Cd含量。进一步的研究发现，Cd诱导的NO通过改变细胞壁多糖的特性增加Cd在根细胞壁上的附着位点并最终促进Cd在根中的累积；（3）克隆获得三七Pn14-3-3基因并构建pCAMBIA1300-35S-Pn14-3-3植物表达载体，转化烟草后获得过表达三七Pn14-3-3基因的转基因烟草。研究结果表明，过表达Pn14-3-3的转基因烟草根中NR活性和NO含量均下降，且Cd含量也显著下降；（4）开展了镁与田间三七提质增效研究。田间试验研究结果表明，镁显著降低三七根中Cd含量达58%，且根中药用成分皂苷的含量也提高18%；（5）本项目还开展了镁提高植物对铝毒胁迫的抗性。研究结果表明，Mg通过抑制铝诱导的NO产生提高三七和拟南芥对铝毒胁迫的抗性；（6）在本项目支持下，共发表SCI论文8篇，培养毕业硕士研究生6名，申请国家发明专利2项。.综上所述，本项目揭示了NR磷酸化及其与14-3-3蛋白互作调控三七根中NO产生和Cd累积的调控机理，完成拟定的研究内容和研究目标。