| Panax is a small genus of the Araliaceae and almost all plants in this genus have a long history of medicinal use.Among of these medicinal plants,ginseng(P.ginseng CA Mey.)is the oldest,most widely and clinical used traditional Chinese herbal medicines which has a huge market demand in the whole world.As a perennial herb of Araliaceae,ginseng can be cultivated for up to twenty or thirty years under simulated wild conditions,and hundreds of years in the wild.It is an extremely rare long-lived herb.Telomere is a complex structure consisting of a simple repetitive DNA sequence and a telomere-binding protein at the end of a chromosome of a eukaryotic organism.In higher organisms,telomeres in cells often attrited as the number of cell divisions increases.When attrited to a certain threshold(Hayflick limit),cells stop dividing into the senescence stage,so telomeres are called 'Molecular clock','Life clock' The telomere sequence in plants has important functions such as maintaining chromosome and genetic stability,affecting the physiological and pathological processes of plants,and closely related to plant longevity.Telomere is a research subject worthy of attention.The telomere length in ginseng is linearly related to the age of ginseng and is the best biomarker for ginseng age identification.Ginsenoside is the most important pharmacologically active ingredient and an important indicator for quality evaluation in ginseng.Ginsenoside contained in soil which has been continuously cultivated for many years has significant autotoxic effect,which makes ginseng have obvious continuous cropping obstacles.Experiments also showed that ginsenoside has a significant effect on the growth and development of ginseng seeds and seedlings.The significant effect of exogenous ginsenosides that extremely narrow distributed in the plant kingdom on the physiological activities of ginseng itself suggests that it may have a certain physiological relationship with the telomere length in ginseng,thereby maintaining the long life-cycle of ginseng.In order to verify the scientific hypothesis that there is a physiological connection between ginsenosides and telomere length,this paper carried out the following work:1.A new Q-PCR method for rapid determination of telomere length was developed and evaluated by the classical TRF method for telomere length determination.For the first time,a method for high-throughput determination of telomere length was established.Firstly,the telomere length of ginseng root was determined by the accepted "gold standard" TRF method for telomere length measurement.Secondly,the Q-PCR method was developed to determine the relative telomere length of the main root of ginseng.According to the existing research,the results of the PCR method were correlated with the TRF method and the correlation between the two methods was good(R2=0.7577).The established PCR method can be used for the rapid determination of telomere length in ginseng.2.The effects of exogenous ginsenosides on the telomere length of ginseng suspension cells were investigated.The results showed that some of the protopanaxadiol type ginsenosides can shorten the telomere length,and some of the protopanaxatriol type ginsenosides can prolong the telomere length.Exogenous ginsenosides have close physiological relationship with telomere length was confirmed for the first time and it's the first time that some of the protopanaxadiol type ginsenosides and some of the protopanaxatriol type ginsenosides may have antagonistic functions in ginseng.Using the method of exogenous addition,six different ginsenosides GS1-GS6 and two plant hormones methyl jasmonate and brassinolide were added to the culture medium of ginseng suspension cells,and ginseng was collected after one day and seven days of culture.The telomere length of different treated ginseng suspension cells was determined by Q-PCR.The results showed that the protopanaxadiol type ginsenosides GS1,GS3 were added to the medium to the ginseng suspension cells for one day and seven days had significantly shortened telomere,the protopanaxatriol type ginsenosides GS2 for one day and GS5 for seven days can significantly prolong the telomeres of ginseng suspension cells.3.The representative protopanaxadiol type ginsenosides and protopanaxatriol type ginsenosides treated suspension cells were selected,and the differences in transcription levels of them were analyzed.A large number of annotated differentially expressed genes were obtained,which provided evidence for the discovery of a variety of potential physiological functions for ginsenosides.Three biological replicate samples of treatment with 50 ?M MJ,GS1,and GS2 for one day were selected for transcriptomics studies.The results showed that MJ as a plant hormone,had the greatest influence on the transcription of ginseng suspension cells.This were consistent to the results of effects of three treatments on telomere length of ginseng suspension cells.The annotation analysis of KEGG database showed that there were more differential genes in plant hormone signal transduction,starch and sucrose metabolism after MJ treatment,and endoplasmic reticulum after ginsenoside GS1 and GS2 treatment.There are more differential genes in the two pathways of protein processing in endoplasmic reticulum and plant-pathogen interaction,indicating that these two treatments are different from MJ treatment,other than through hormone signaling pathways but through pathogen pathways to affect ginseng suspension cells.4.The regeneration system and genetic transformation system of ginseng were established,and the overexpression and RNA interference of key enzyme genes of ginsenoside biosynthesis were conducted.The physiological relationship between endogenous ginsenoside and telomere length was discussed for the first time.The ginseng callus was induced by dark culture of ginseng explants from various sources,and then transferred to a differentiation medium for light culture to obtain cluster buds.The buds were transferred to rooting medium,and regeneration seedlings was obtained by light culture.The key enzyme gene of ginsenoside biosynthesis was cloned by cDNA reverse transcription of ginseng root.The corresponding expression vector was constructed by Gateway cloning technology.The vector was transformed into Agrobacterium tumefaciens EHA105 and Agrobacterium rhizogenes C58C1 by freeze-thaw method,and ginseng was infested by leaf disc method with the two kind of Agrobacterium.The obtained vector was wrapped on the surface of gold powder and directly injected into the ginseng callus by bombardment with a gene gun.The construction of ginseng genetic transformation system was explored by both Agrobacterium infection method and gene gun method. |
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