Response Of Ginsenosides Synthesis And Its Key Enzyme Genes Expression To Ecological Factors In Different Growth Period

Panax ginseng is one of the most important cultivated medicinal plants in Jilin province.Synthesis of ginsenosides were co-regulated by the environmental of ginseng growth and the key enzyme genes.Try to analyze the relationship between the key enzyme genes expression and the content of ginsenosides with environmental of ginseng growth,to elucidate the physiological and ecological mechanism of ginsenoside synthesis,which can provide theoretical support for improving the quality of Radix Gineng.The expression levels of key enzyme genes in roots,stems and leaves of ginseng in the different growth period and 8 kinds of monomer ginsenosides content were determined,and the relationship between ecological factors and the expression of key enzyme genes and ginsenosides content was analyzed in this experiment.The main results were as follows:1.Four-year-old ginseng roots were used as test materials,to establish a standard method of real-time fluorescent quantitative RT-PCR by ginseng Cyclophilin reference gene.The method to detect Cyclophilin gene had a good linear relationship in a wide range from 1.11×104 to 1.11×109copies·μL-1(R2 = 0.9998);The melting curve showed a single peak;The coefficient of variation(CV)of five different concentration of positive plasmids was 0.33% to 2.65% in intra-assay and 2.81% to 4.22% in inter-assay respectively.This method had the advantages of rapid,accurate with good repeatability,high sensitivity,simple operation,short time,use the ginseng Cyclophilin gene as a reference gene provided a theoretical basis for ginsenoside biosynthesis related key enzyme quantitative expression analysis.2.Four-year-old ginseng roots were used as test materials,the expression of key enzyme genes(HMGR,FPS,SS,SE,DS,β-AS,CYP82D47,CYP716A47)in the biosynthesis of ginsenosides of roots,stems and leaves in different growth period was determined by real-time quantitative PCR.The period of leaf-expansion(May 30th)and fruit(from preliminary period of green fruit to the red fruit period,from July 3rd to August 5th)is the active period of ginseng HMGR,FPS,SS,SE,DS,CYP716A47 gene expression,the key enzyme gene expression levels is higher at this time,it is the important period to control the ginsenoside synthesis.Ginseng β-AS gene was mainly expressed in roots,the expression levels in stems and leaves were very low.The expression of ginseng CYP82D47 gene in stems and leaves were gradually increased,but almost no expression in roots.The expression of the key enzyme genes involved in the synthesis of ginseng saponins,which were correlated with each other,and increased with each other,showed a high degree of correlation in roots,stems and leaves.There was a positive correlation between the expression of key enzyme genes and the accumulation of ginsenosides in ginseng roots and stems,wihle the expression of key enzyme genes in leaves showed a negative correlation with the accumulation of ginsenosides.The expression of key enzyme genes in ginseng were significant tissue differences.3.Four-year-old ginseng roots were used as test materials,determination of the content of 8 ginsenosides in different tissues of ginseng by HPLC.The content of total saponins in ginseng leaves > roots > stems,ginsenoside Rg1 and Re were higher in different tissues,while ginsenoside Rd had higher content in leaves.The peak distribution of protopanaxatriol Rg1,Re and Rf in ginseng roots was in the early stage of ginseng growth,while the peak distribution of protopanaxadiol Rb1,Rc,Rb2,Rb3,Rd was in the middle and late stage of ginseng growth.The peak distribution of 8 kinds of monomer saponins in ginseng stems was mainly in the early stage of ginseng growth(from May 30 th to June 22nd).The peak distribution of 8 kinds of monomer saponins in ginseng leaves was mainly in the post fruiting stage Ⅰ.The distribution of 8 ginsenosides in ginseng leaves,stems,roots is not the same.4.The correlation of the key enzyme genes expression levels with ecological factors(temperature,Photosynthetic active radiation,Soil water potential,Rainfall,Relative humidity)and ginsenoside contents in ginseng roots,stems and leaaves in different growth stages was analyzed.Ecological factors and the expression levels of the key enzyme genes involved in the synthesis of ginsenosides had important effects on the synthesis of ginsenosides.The high temperature had a significant inhibitory effect on the synthesis of most of the monomer saponins in ginseng leaves and protopanaxadiol in roots,while the temperature had a positive effect on the expression levels of key enzyme genes in roots and stems,but the expression levels of key enzyme genes in leaves showed a weak inhibitory effect.PAR could inhibit the accumulation of ginsenosides and the expression levels of key enzyme genes in leaves,however,it promoted the accumulation of ginsenosides and the expression levels of key enzyme genes in roots and stems.Soil water potential significantly increased the expression levels of key enzyme genes in roots,stems and leaves,but the high soil water potential affected the accumulation of ginsenosides in the roots and leaves.The relative humidity can significantly influence the accumulation of ginsenosides in leaves.Because rainfall had no direct effect on the ginseng plants,therefore rainfall on ginsenosides did not have significant influence.Grey correlation analysis results showed that the major ecological factors that influenced ginsenosides content in ginseng roots were temperature,PAR and relative humidity;the major ecological factors that influenced ginsenosides content in ginseng stems were PAR and relative humidity,the secondary ecological factors that influenced ginsenosides content in ginseng stems were temperature;the major ecological factors that influenced ginsenosides content in ginseng leaves were relative humidity and soil water potential,the secondary ecological factors that influenced ginsenosides content in ginseng leaves were temperature and PAR;the grey correlation between the expression of the key enzyme genes with the content of ginsenosides is less than ecological factors with the content of ginsenosides,under the guidance of ecological factors,the expression of the key enzyme genes regulate the synthesis and accumulation of ginsenosides.