| There are 18 species in the genus of Panax. Among them, only three valuable medicinal herbs(Panax ginseng, Panax quinquefolius and Panax notoginseng) have received considerable interest due to their extensive application in clinical therapy, healthcare products, and as foods and food additives world-wide. By contrast, other Panax species attracted little attention because of their limited distribution area, less wide application or relatively low quantity. Panax vietnamensis(PV) and Panax stipuleanatus(PS) and Panax notoginseng(PN) are mainly distributed in Southeast Asia. These three medicinal plants which had similar shape features and geographical distributions are often confused used by locals in southwestern China and northern Vietnam. In this study, chemical fingerprints analysis combined with chemometric approaches, GC-MS, molecular marker SCoT technology and DNA barcode based on ITS had been used to establish and compare the chemical fingerprint, volatile composition and genetic diversity among the three species. Moreover, PN was called as ―the king of ginseng‖ due to its high content of saponins. The main physiological active compositions were saponins compounds in PN. At the same time, it contains amino acids, polysaccharides, flavonoids, alkyne alcohols and volatile oil and other compounds. To obtain more knowledge on the secondary metabolic pathways of PN, the composition of seeds, the accumulating characteristics of saponins and the key enzyme genes of saponin synthesis were investigated in the study. The main content was as follows:1. In the present study, an optimized and reliable HPLC method with more appropriate equipments was developed to identify and distinguish the three Panax. The Photodiode Array Detector(PAD) and SunFire column were the better choice to establish the chromatographic fingerprint. Based on the HPLC-PAD method, the chemical chromatographic fingerprints of the three Panax medicinal species were established, respectively. By comparing the chromatograms, the three Panax genus species could be distinguished easily using the 22 characteristic peaks. Chemometric methods were used to evaluate the minor differences among the chromatograms. The results indicated PN revealed more diversity in the content of saponins and the growth regions and fertilizer treatments had little influence on the variety of the saponins. Besides, the chemical compositions revealed higher similarity between PV and PS than PN through the coefficient of similarity.2. GC-MS was successfully employed to analyze and compare the volatile compounds from the three Panax species. The results indicated the numbers(about 70) of the volatile oils compounds in the three Panax species were similar while the contents of every compound were great different. 120 compounds were identified from the three species. 26 compounds were all contained among the three species. By comparing the compounds, it is clear that the volatile oils of PN, PV and PS were mainly composed by the falcarinol which had well biological activity. Moreover, PN contained a high andrographolide(38.35%) proportion. The results of HCA indicated that PV and PS exhibited high similar based on the high content compositions of their volatile oils.3. To evaluate the genetic diversity of PN and its two related species(PV and PS), Start Codon Targeted(SCoT) marker system were first used in the study. 16 primers were selected and SCoT markers provided a high polymorphism rate of 89.47% which was higher than the 58% in previous study. Analysis of molecular variance(AMOVA) showed 61.34% of variation within the populations, whereas 38.66% variation was recorded among the populations. The results of the internal transcribed spacer(ITS) DNA barcode marker indicated that only one variation site was detected within PN and 38 sites(5.9%) were variable among the three species. Based on the two markers, cluster analysis, the homology tree and the phylogenetic tree all revealed the same conclusion that PV showed a closer phylogenetic relationship with PN than PS.4. The Crude fat of seed in PN was extracted by Soxhlet fat extraction instrument and the seed is rich of fat(46.35%) which is comparable with both contents in sunflower and rape seed. According the GC, Seven fatty acid compositions were determined and abundant in unsaturated fatty acid(99.56%), containing 95.71% oleic acid. Kjeldahl method and the Automatic Amino Acid Analyzer were used to detect the seed and the content of total protein is 23.90%, which contains 17 kinds of amino acids. Except for valine, the essential amino acid scores were higher than the FAO/WHO mode index, which indicates that amino acids in PN seed can meet the demand of human body with intake balance and high nutritional value. These nutritional compositions indicate that PN seed has the potentiality to be exploited as human edible oil, industrial use, new medicine or healthcare products for diabetes patients due to low sugar content.5. An improved, simple, and reliable HPLC method was developed to simultaneously quantify five major saponins extracted from P. notoginseng. The best method for saponins extraction was that 0.2 g samples was soaked by 8 mL 70% aqueous methanol with overnight immersion, and extracted by ultrasonic bath. Using the optimized method, the accumulation properties of the five main saponins during three years were studied in the different parts of PN. During three growing seasons, September in the third year was the best harvest time due to the highest content of five major saponins at that time. Removing the flower bud in the third year would significantly increase the content of saponins in root of PN.6. Based on the transcriptome data of PN, four genes of key enzymes in saponin synthesis- farnesyl pyrophosphate synthase(FPPS), squalene synthase(SS), squalene epoxidase(SE) and dammarenediol synthase(DS) were cloned from the PN. Their nucleotide sequence contained open reading frame(ORF) of 1029 bp, 1248 bp, 1614 bp and 2310 bp, and coded 342, 415, 537 and 769 amino acids respectively. According the SMART, there were two, four and one transmembrane regions in the SS, SE and DS, respectively. Besides, one low complexity region was found in the SE.7. To examine the subcellular localization of the four key enzymes in saponin synthesis, the ORF of their nucleotide sequences were fused to the GFP reporter gene under control of the CaMV 35 S promoter. Under the gene gun bombardment, the recombinant plasmids and the GFP alone were expressed in the onion epidermal cells, respectively. The results showed that SE and DS were mainly located in cytomembrane and its surrounding, FPPS and SS were located both in cytoplasm and cytomembrane.8. Prokaryotic expression vector of the four target genes were constructed and the recombinant plasmids were introduced into prokaryotic expression strains E.coli Rosetta. After induced with IPTG by different times, the SDS-PAGE was used to detect the expression of protein. The results indicated that FPPS, SS and DS were strongly expressed in the expression system of pMAL. Under the same condition of IPTG, the maximum expression of FPPS was in the induction of 15 h, and SS and DS was in the induction of 8 h. To determine the induced proteins were soluble protein or inclusions, ultrahigh pressure continuous flow cell crusher at low temperature was used to crush the bacteria. The results indicated that FPPS and SS were soluble protein, and DS was inclusion based on the expression system of pMAL. |
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