人参叶片中人参皂苷含量、关键酶基因表达和生态因子的相关性分析

摘要

[目的]明确人参皂苷生物合成的生理生态机制,揭示生态因子和遗传因素与药材质量的关系.[方法]以4年生不同生长时期的人参叶为试验材料,用HPLC法测定人参叶中8种单体人参皂苷(Rg1、Re、Rf、Rb1、Rb2、Rb3、Rc和Rd)含量;实时荧光定量PCR法测定7个参与人参皂苷合成的关键酶基因(HMGR、FPS、SS、SE、DS、β-AS和CYP716A47)的表达量;通过相关性和灰色关联度分析生态因子和人参皂苷合成关键酶表达对人参叶中皂苷合成和积累的影响.[结果]7月13日—9月29日人参皂苷合成关键酶基因表达活跃,各个关键酶之间有协同增减的趋势;人参叶中单体皂苷含量最高的是Re和Rg1,在果后参根生长期(8月31日—9月13日)分别达到最大值60.30和39.38 mg·g–1;温度、光合有效辐射、土壤水势、相对湿度与叶中人参皂苷含量显著相关(P<0.05);人参叶中HMGR基因的表达与Rb2含量显著负相关(P<0.05),SS基因的表达与Rg1、Re含量显著负相关(P<0.05), β-AS基因的表达分别与Rc、Rb2含量呈显著(P<0.05)、极显著(P<0.01)负相关;温度、光合有效辐射、土壤水势和相对湿度与人参皂苷含量灰色关联度较高,达到0.7279~0.8711.[结论]在生态因子调控下,人参皂苷合成关键酶基因的表达影响人参皂苷的合成与积累.%[Objective]To clarify the physiological and ecological mechanisms of ginsenoside biosynthesis, and reveal the relationship between ecdogical/genetic factors and quality of medicinal materials.[Method]Cultivated four-year-old Panax ginseng leaves at different growth times were used as materials.The contents of eight ginsenosides (Rg1、Re、Rf、Rb1、Rb2、Rb3、Rc、Rd) in leaves were detected by HPLC. The expression of seven key enzyme genes (HMGR, FPS, SS, SE, DS, β-AS, CYP716A47) involved in ginsenoside biosynthesis in ginseng leaves were determined by real-time quantitative fluorescent PCR. The effect of ecological factors and the expression of key enzyme genes on the synthesis and accumulation of ginsenosides in P.ginseng leaves were investigated by correlation and grey relational analysis.[Result]The expression of key enzyme genes involved in ginsenoside synthesis was active from July 13th to September 29th, and the expression of different genes synergistically increased or decreased. The contents of ginsenoside Re and Rg1in P.ginseng leaves were the highest,reached the maximum of 60.30 and 39.38 mg·g–1respectively in the growth period of ginseng root from August 31st to September 13th. Temperature, photosynthetic active radiation (PAR), soil water potential and relative humidity all had significant correlation with ginsenoside contents in P. ginseng leaves(P<0.05).HMGR expression in P.ginseng leaves was negatively correlated with Rb2content (P<0.05),SS expression was negatively correlated with Rg1or Re content(P<0.05),and β-AS expression was negatively correlated with Rc content (P<0.05) and Rb2content (P<0.01). Grey correlation analysis results showed that the major ecological factors that influenced ginsenoside contents in P.ginseng leaves were temperature, PAR, soil water potential and relative humidity, with their grey correlation values ranging from 0.727 9 to 0.871 1.[Conclusion]Under the regulation and control of ecological factors, the synthesis and accumulation of ginsenosides were influenced by the expression of involved key enzyme genes.