| Panax ginseng C.A.Meyer is one of the most important medicinal plants that belongs to Araliaceae.Ginsenosides,the main medicinal bioactive compounds of Panax ginseng,is an important indicator for evaluating the quality of ginseng medicinal materials.Our research group analyzed the big data of the correlation between ginsenosides content and ecological factors such as climate,soil and altitude from 116 cultivated plots in 15 counties and cities around the source areas of Changbai mountains and studied relevant scholars’ research,we concluded that temperature is an important ecological factor affecting ginsenosides content,and appropriate low temperature is conducive to the synthesis and accumulation of ginsenosides.However,there are few studies on the effect of low temperature on the expression of key enzyme genes in ginsenoside biosynthesis pathway,and the molecular mechanism of physiological and ecological response under low temperature is still unclear.Therefore,this study,based on the perspective of plant physiology,ecology and molecular ecology,took potted ginseng and its callus as the research object,defined the sensitive temperature affecting ginsenoside synthesis through temperature regulation,and understood the response law of ginsenoside synthesis and accumulation to temperature regulation.Based on the above,by means of potted plant,we studied the dynamic change of ginseng saponin content under the condition of low temperature,combined it with ginseng resistant physiological（antioxidant enzymes,osmoregulation substance and endogenous hormone,etc.）and ginseng saponin synthesis way key enzyme gene expression.By using the transcriptome sequencing and microRNAs sequencing technology,we made a comprehensive analysis on ginseng saponin biosynthesis in transcription level and the low temperature control mode at the transcription level,as well as an interpretation of ginseng on the molecular mechanism of low temperature regulation of the physiological ecological response,aimed at providing theory support for promoted ginseng herbs quality and ecological control of technology innovation.The main findings are as followed:（1）By regulating the temperature of ginseng callus,the low temperature response of ginsenoside synthesis and accumulation was revealed,and it was found that low temperature had a short-term promoting effect on ginsenoside synthesis and accumulation.Studies on potted ginseng showed that under the short-term low-temperature regulation,the contents of ppd-ginsenosides in the taproot（phloem and xylem）,fibrous roots and fibrous roots were significantly increased,and the xylem of the taproot was the sensitive part in response to low temperature.It was found that the change of temperature had a significant effect on the PPD/PPT value of ginsenoside components,and low temperature was an important restriction factor to regulate the accumulation of ginsenoside synthesis.（2）Correlation analysis of ginsenosides and physiological indexes of resistance in potted ginseng and its callus showed that contents of antioxidant enzymes（CAT,POD,SOD）,osmotic regulatory substances（Pro,SS,SP）,endogenous hormones（ABA,SA,JA）and ginsenosides showed convergence adaptation to low temperature.The antioxidant enzyme activity,osmotic regulatory substance content,endogenous hormone content and ginsenoside content of ginseng callus showed an increasing trend under short-time and low-temperature conditions.The antioxidant enzyme activity,osmotic regulatory substance content and ginsenoside content of potted ginseng showed an increasing trend,but the degree of change was less than that of ginseng callus.It was found that ginsenoside synthesis and physiological indexes of resistance were convergent to low temperature,which may be that ginsenoside secondary metabolism is the compensation mechanism for ginseng to adapt to low temperature.（3）The correlation between HMGR2,FPS,SS1,SE1,DS-Ⅱ,PNY1,CYP716A52v2,CYP716A53v2 and CYP716A47 gene expression and ginsenosides under low temperature regulation showed that key enzyme genes responded to low temperature in different degrees,short low temperature regulating the biosynthesis of ginsenosides key enzyme genes in a pathway,HMGR2,SS1 and DS-Ⅱ rised significantly,PNY1 genetic level of low temperature response is not obvious,FPS,SE1,CYP716A53v2 and CYP716A52v2 showed different expression patterns in different tissues of ginseng.The expression of key enzyme genes was closely related and had the same expression pattern as the content of saponins,indicating that multiple key enzyme genes in the synthesis pathway coordinated regulation and guidance of ginsenoside biosynthesis.（4）Sequencing of the ginseng transcriptome under low temperature regulation revealed that a large number of differential genes were annotated into the metabolic pathways such as plant hormone signaling pathway,redox process,secondary metabolite biosynthesis,and transcription factor activity.Low temperature regulation promotes the up-regulation of NCED,ZEP and SDR gene expression in the ABA synthesis pathway,suggesting that low temperature regulation enhances the synthesis of secondary metabolites by activating ABA by stimulating the corresponding transcription factors.The transcription factors MYB,bZIP,AP2 and ARF were significantly up-regulated in the T4 vs C4 group,which showed the same trend as the saponin content in ginseng,and could be used as the main candidate transcription factor in response to low temperature.（5）A total of 482 new miRNAs and 265 conserved miRNAs were identified by sequencing ginseng miRNAs under low temperature regulation.133 miRNAs were specifically expressed in the T4 group,and 103 miRNAs were specifically expressed in the C4 group,and 16 differentially expressed miRNAs were identified,of which pgi-MIR6135h-p31ss22TC and pgi-MIR6135d-p31ss23CT were associated with the target gene DNA-3-methyladenine glycosylase has a negative correlation,this indicated that under the regulation of low temperature,two miRNA genes regulated ginsenoside secondary metabolism process by degradation methylase,and promoted ginsenoside biosynthesis. |
