云南道地药材三七皂苷和黄酮产量对土壤As的响应及其机理研究

As contents in Panax notoginseng (Burk) F.H.Chen exceeding the national standard value,which would influence in the security of medical plant Panax notoginseng, were reported. Panax notoginseng as geoherbs of Yunnan Province would be chosen as research materials. The field investigation and simulation trials would be conducted with theory and methods of environmental geology and mocular biology. The aims are: (1) to understand contents of As, total saponins, singal saponins R1, Rg1 and Rb1, flavonoids, biomass, activities of squalene synthase (SS), phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) in main roots, fibrous roots, stem, leaves, flowers and fruits of two-year-old and three-year-old Panax notoginseng with different soil As contents in original and mainly production areas of Panax notoginseng with high soil background value of As; (2) to analysis the responses of saponins (saponins contents × biomass), flavonoid (flavonoid contents × biomass) yields to soil As; and (3) to clarify the enzymological and molecular mechanisms of responses of saponins and flavonoid yields to soil As based on activities of key synthases and its genes expression. That would be of important theory and practice roles for increase in yields of pharmacological compounds of Panax notoginseng and promotion industrial development and ecological security of Panax notoginseng.

在三七砷超标威胁到三七药物质量安全的背景下，以云南道地药材三七为研究对象，应用环境地学和分子生物学理论，采用大田调查和田间试验相结合的方法，研究三七原产地和主产区土壤As高背景区，不同土壤As含量水平下，三七二年生和三年生植株主根、须根、茎、叶、花或果实中As含量、生物量、总皂苷含量、单体皂苷R1、Rg1和Rb1含量、黄酮含量、三七鲨烯合酶活性、苯丙氨酸解氨酶活性和查尔酮合酶活性；分析三七皂苷产量(皂苷含量×生物量)、黄酮产量(黄酮含量×生物量)对土壤As的响应；通过皂苷和黄酮合成关键酶的活性及基因的转录表达，探讨三七皂苷产量、黄酮产量对土壤As响应的酶学机理和分子机理。对于提高三七药用成分产量和安全性，促进三七产业发展和生态安全具有重要的理论和实践意义。

通过大田调查和模拟试验，研究三七皂苷和黄酮含量对砷胁迫的响应及其酶学和分子机制。结果表明：文山州76.67%的三七种植土壤总含量达到国家土壤环境质量的二级标准。三七各部位中As含量符合优质三七砷的限量标准(≤2 mg•kg-1)。文山州三七剪口中总皂苷（R1+ Rg1+ Rb1）和主根黄酮平均含量分别为13.51%和3.59 mg•g-1。连续2年As胁迫处理，三年生三七各部位砷含量随着砷处理浓度增加而增加。须根中As含量大于剪口、主根。260 mg•kg-1As处理下须根砷含量超过了三七砷含量标准。砷胁迫条件下，根系分泌物中柠檬酸和总有机酸含量随砷处理浓度的增加而增加。三年生三七主根中总皂苷含量随砷处理浓度的增加而增加。三年生三七各部位黄酮含量均随砷处理浓度的增加而降低。.从酶学机制来看，80-140 mg•kg-1 As处理时，在剪口、主根和须根中三七鲨烯合酶活性均表现出显著的增加，260 mg•kg-1 As处理时，导致总皂苷含量和三七鲨烯合酶的活性显著降低。As ≤ 200 mg kg-1处理，须根查尔酮合酶活性提高，苯丙氨酸解氨酶活性降低。从蛋白质组学机制来看，140 mg•kg-1As处理，下调的蛋白质为磷酸核酮糖激酶、热激蛋白、NAD(P)-结合酶、单脱氢抗坏血酸还原酶和细胞色素b6-f复合物铁硫亚基。上调的蛋白质为CDC27家族蛋白、异黄酮还原酶、苹果酸脱氢酶和谷氨酰胺合成酶。从分子机制来看，砷处理下, PAL 基因、CHS基因 和CHI基因表达量下降。细胞色素基因(CYP71D444和CYP73A100)和糖基转移酶基因（UGTPg25)的表达下调是皂苷代谢的限制基因。三七生长和皂苷、黄酮含量对砷的耐受阈值为140 mg•kg-1。.创新点：首次报道了F3H 和FLS是黄酮代谢最关键的限速酶，槲皮素与山萘酚是三七黄酮的主要组成成分。皂苷累积与SE (PnSE2)和细胞色素P450 (CYP716A52v2 和CYP450A47) 基因的表达上调有关，皂苷的异质性受到细胞色素P450 (CYP71D444 and CYP73A100) 和糖基转移酶 (UGTPg25)基因表达的影响。.项目发表论文18篇，其中SCI论文4篇，EI论文2篇。出版专著《重金属污染生态与生态修复》1部。获获得的新项目2项，培养硕士研究生4名，国际会议大会交流和分会场报告4次