Effect Of Ecological Factors On Ginsenoside Content Of Panax Ginseng And The Expression Of Key Biosynthesis Enzymes Genes

Panax ginseng is one of the most important medicinal plants that belongs to Araliaceae, its application has a long history. ChangbaiMountains in Jilin province is main producing areas,medicinal materials, a lot of drugs and health care products of P. ginseng that have been supplied the country and exported to many countries and regions. The deficiency of study on main medicinal component biosynthesis and accumulation of ginsenoside has restricted the innovation of medical material regulation technique. The correlation research on biosynthesis and accumulation of ginsenoside and genetic expressions of key enzyme genes and ecological factors from the point of view of ecology is significant in illuminating ecological control mechanism and dominant factor of ginsenoside synthesis, driving the scientific and technological progress for medicinal materials quality control techniques of P. ginseng.In this paper, the cultivated P. ginseng in Jilin province were taken as the research object,the sampling areas covers 15 counties and cities of geo-authentic habitats of P. ginseng, 127 sample plots were set, which were located 40°58′1.2″~44°26′54.6″ north latitude and125°20′43.8″~130°51′24.1″ east longitude, crossing 3.4 latitudes and 5.5 longitudes. The altitude was from 96 m to 1450 m, the altitude difference was 1354 m. The 13000 original data were obtained in this paper, including the content of total saponins and eight kinds of monomer saponins of P. ginseng, key enzyme gene expression quantity of FPS, DS, β-AS and CYP716A47 in biosynthetic pathway of ginsenoside, soil pH, conductivity, salinity content and key soil elements and so on. Based on GIS geographical information technology, 127 sample status and9 meteorological factor were obtained by multiple linear regression of DEMand applying of spatial interpolation function of the ArcGIS software. The cultivated P. ginseng sample area of continuous cultivation for five years of Laoling in Fusong County was continuous observed for24 hours in whole growing season by microclimate positioning observation system. The big data analysis for correlation between ginsenoside contents, genetic expressions of key enzyme genes of ginsenoside biosynthesis and ecological factors such as climate, soil, altitude and so on was studied on the macro spatial-scale and micro time-scale. The effect of ecological factors on medical material quantity of P. ginseng were illuminated, the ecological control strategy and relevant dominant factors on biosynthesis and accumulation of ginsenoside were proposed. This paper can provide scientific basis of ecological regulation for boosting medicinal material quality of P. ginseng. The main research results were as follows:(1) The ultrasonic-assisted extraction of Rb1、Rb2、Rb3、Rc、Rd、Rg1、Re and Rf fromginseng was established, the extraction rate was 1.42 and 5.57 times as much as H2 O and ethanol extraction method. The optimum processing conditions was taken [C3MIM]Br(0.3mol/L)as dissolvent, material-to-liquid ratio(g/mL) is 1:10 and extraction 20 min by ultrasonic.(2) The genes encoding key enzymes of FPS、DS、β-AS and CYP716A47 for ginsenoside synthesis were cloned successfully. The real-time PCR method for the detection of GAPDH gene was established. The experimental results showed that the method was sensitive, specific,fast and strong repeatly, etc. The results of this study will provide basis for analyzing the quantitative expressions of ginseng functional genes.(3) The quality of P. ginseng from 15 major producing areas in Jilin province was conformed with the stand of China Pharmacopeia. The contents of Rb1, Re+Rg1 respectively were 2.10-5.90, 2.68-5.10 times as much as national standards. The contents of total saponins,Rb1, Rc, Rg1 and Rf increased hardly with cultivated time, among them Rb1 and Rg1 were the biggest increased, they could increase to 1.1796% and 1.0510% of the quality of ginseng, which showed the prolonging of cultivated time was advantageous for synthesis and accumulation of Rb1 and Rg1. Cluster analysis showed the distribution of different quality has gradually converted from flake to strip, and the strip regionalism was gradiently distributes around the ChangBaimountain, the reason need a further in-depth study.(4) The altitude has significant effect on the contents of total ginsenosides, cultivated areas in 600-1000 m above sea level were advantageous for accumulation of total ginsenosides. The relationship between 8 kinds of monomer saponins and altitude changes was complicated, the distribution of Rb1 and Rc was bimodal, and in high altitude regions is higher than low altitude regions; the distribution of Rg1, Re, Rf, Rb2, Rb3 and Rd was single peak type, the peaks of Rg1,Re, Rf and Rb2 has appeared in altitude 600-800 m, the peaks of Rb3 and Rd has appeared in altitude 800-1000 m. Significant analysis of monomer saponin content between different altitude showed that the significant difference of Rb1, Rg1 and Re was unconspicuous, especially Rb1 showed no significant difference between altitude 300-1000 m.(5) The regression coefficients and weight coefficients of VIP between climate factors and total saponins and eight kinds of monomer saponins contents of P. ginseng were analyzed by using partial least squares method. The research showed that ginseng medicinal materials quality was significantly negative correlation with temperature changes, and was significantly positive correlation with hours of sunshine and the annual precipitation. The results indicated that a long time sunshine, high annual precipitation and low temperature conditions were benefit to the synthesis and accumulation of ginsenoside. The saponin contents of P. ginseng roots which were different ages were different respond to the climate differences, for example, the hours ofsunshine may be even more important for the contents of the four-year old P. ginseng, and a low temperature condition was more important for the contents of five-year or six-year old P.ginseng, which the existing data is difficult to explain and still need to be further studied.(6) The partial correlation coefficient and decision index of path analysis between the soil factors and the total saponins, and eight kinds of monomer saponin of P. ginseng were analyzed by multiple stepwise regression. The results showed that the herbs quality of P. ginseng was significantly positive with soil pH, which indicated that soil acidification inhibited ginsenoside synthesis; The soil K, Ca and Mg elements content were significantly positively related with ginseng herbs quality, and the content of Cu, Mn, Zn in soil had promoted the monomer saponin synthesis of P. ginseng, but the increase of Na content had disadvantage of ginsenoside synthesis.(7) The genes expression of FPS, DS, β-AS and CYP716A47 showed that the four genes were also expressed in leaves, green fruits, red fruits, the root growth after fruit periods and root,stem, leaf and fruit. The expression quantity of β-AS obviously increased in green fruit period,and has been on a rally, it achieve relatively high level at the root growth period after fruit period, its key expression parts was in the root, which was significantly higher than other parts.The expression peak of DS appeared in the root growth period after fruit period, which was significantly higher than other periods, and the key expression location at leaves, and significantly higher than other parts. The expression peak of FPS appeared in the red period,which was significantly higher than other periods, its key expression parts were the root and leaves. The FPS expression in leaves was comparative advantage. The CYP716A47 expression peak also appeared in the red period, which was significantly higher than other periods, the key expression parts were leaves and fruit, The CYP716A47 expression in leaves was comparative advantage. The expression of DS significantly increased the Rb2, Rb3, Rc, Rd contents, while the expression of CYP716A47 significantly increased the content of Re, the expressions of both were significantly increased the contents of total saponins.(8) The correlation study between ecological factors and FPS, DS, β-AS and CYP716A47 gene expression showed that the relative humidity and soil water can increase the FPS, DS, β-AS and CYP716A47 genes expression significantly, the expression of the DS and β-AS and CYP716A47 of downstream genes in ginsenoside synthesis pathways can significantly increase the total saponins synthesis and accumulation, the FPS expression of the midstream in ginsenoside synthesis pathways did not significantly affect total saponin content. The study showed that the key parts genes of ginsenoside synthesis were downstream genes in moisture factor regulation. There have significant negative correlation between DS expression andaltitude, the optimum range from 700 m to 900 m. In the four genes, DS was sensitive to changes of ecological factors, which reflected that the DS is likely to be important ecological control genes of ginsenoside synthesis.