有效磷水平对三七砷吸收、转运的影响及调控机制研究

Panax notoginseng planted in Wenshan Prefecture Yunnan province is not only the most famous genuine Chinese drug and the most advantages biological resources, but also the unique valuable Chinese herbal medicine in China. While panax notoginseng were serious ruined by arsenic problem, which limits its quality and safety. In the present project，Wenshan Panax notoginseng was used as research materials, the effect of available phosphorus on the uptake and transport of arsenic was investigated, as well as the different concentrations of phosphorus and arsenic on the growth and saponins biosynthesis of Panax notoginseng. Moreover, Pht1 genes were cloned, and their regulation mechanism in relation to arsenic uptake and transport was revealed through a series of experiments, including subcellular localization analysis, yeast function complementary verification, tobacco over-expression, spatial and temporal expression profiles and promoter activity analysis. The results described in this project through improving effective phosphorus level to inhibit the uptake and transport of arsenic would be not only useful for panax notoginseng’s food and drug safety through reducing arsenic uptake, but also for improving and increasing the yield and quality of panax notoginseng, which has double actual significance for cultivation and production of panax notoginseng. In addition, the understanding of the functional and regulation mechanism of Pht1 genes associated with arsenic uptake and transport may provide a theoretical basis and experimental instruction to repair arsenic pollution.

文山三七是云南省最为著名的道地药材和最具特色优势的生物资源，也是我国特有的名贵中草药。但三七砷超标问题突出，严重损害了其品质和安全。本项目拟以云南文山三七为研究材料，明确有效磷水平对三七砷吸收、转运的影响，不同磷、砷浓度与三七生长发育、皂苷合成之间的关系；并进一步克隆高亲和磷酸盐转运蛋白（Pht1）基因，通过亚细胞定位、酵母功能互补、烟草超表达、Pht1基因时空表达、启动子活性分析等实验，从转录水平上阐明不同磷水平下，Pht1基因响应三七砷吸收、转运的作用机理及调控机制。本项目通过改善有效磷水平来抑制三七砷的吸收，不仅可以有效减轻或解决三七砷超标的难题、保障三七的食用及用药安全性，还可以因磷施肥的改善而提高三七的产量和品质，对三七的种植、生产具有双重实践意义。此外，明确Pht1在三七砷吸收、转运过程中的作用机理和调控机制还可为砷污染地区治理和修复提供理论基础和实践指导。

文山三七是云南省最为著名的道地药材和最具特色优势的生物资源，也是我国传统名贵中草药。文山三七砷超标问题突出，严重损害了其品质和使用安全性。调查显示，高砷土壤背景是造成三七砷超标的主要途径。本项目以文山三七为研究材料，通过调整栽培基质中不同磷、砷浓度，明确了有效磷水平对三七砷吸收、转运及抗氧化系统和皂苷积累的影响；通过转录组测序，明确响应低磷、砷胁迫的代谢通路；同时通过基因克隆、生物信息学分析、qPCR实验、目标基因异源表达和功能验证，明确了高亲和磷酸盐转运蛋白（Pht1）基因的表达模式及磷、砷交互处理下的作用机制。结果显示，在相同砷浓度下，根、茎、叶中砷含量随磷浓度的增加而降低；且适磷（136 mg/L）处理可以促进三七皂苷R1、人参皂苷Rg1、Rb1及总皂苷含量的积累和三七生物量的增加。相同磷水平下，高砷胁迫会提高三七根中SOD、POD、CAT酶活及抗氧化物质MDA、GSH、Pro含量。根据转录组测序结果，通过GO、KOG功能聚类和KEGG代谢途径分析发现，差异基因主要分布在代谢通路和细胞合成部分；代谢通路差异基因主要富集在核糖体合成、PI3K−Akt信号通路等；分子功能的差异基因主要为各种酶的催化活性；其中三七皂苷合成关键酶CYP450、FPS、CAS家族基因在磷砷交互处理下变化差异较大。此外，获得了11条含完整ORF的磷酸盐转运蛋白（phosphate transporter, Pht）基因，分属于Pht1、Pht2、Pht3和PhO4个亚族，并重点研究了PnPht1;1、PnPht1;2和PnPht1;3组织表达特性和磷、砷胁迫下的作用机制。PnPht1;1、PnPht1;2和PnPht1;3均编码高亲和磷酸盐转运蛋白，可响应低磷和砷胁迫而显著上调表达，并可增强转化子的低磷吸收能力和砷胁迫能力；亚细胞定位显示，PnPht1;1、PnPht1;2和PnPht1;3为细胞质膜蛋白，通过改变氢离子浓度和ATP耗能来完成对磷酸盐和砷酸盐离子的吸收和转运。本项目通过改善有效磷水平来抑制三七砷的吸收，是解决三七砷超标的有效途径之一，对三七的种植、生产具有重要的实践意义。