| Panax japonicus C.A. Meyer, a perennial herb, belongs to the family of Araliaceae. As one of the precious Chinese herbal medicine in seven categories, it is pharmacologically values of promoting blood flow of P. notoginseng and strengthening tonics of north P.ginseng. P. japonicus also possesses lots of bioactivities, including antiinflammatory and analgesia, immunoloregulation, anti-MI, anti-Bglu, anti-aging, anti-tiredness and anticancer. In the past years, P. japonicus was considerably applied in clinic because of its pharmacologic function. As a consequence, the natural resources were severely destroyed. Therefore, only depending on the production of wild crude drug, is it difficult to meet the needs of the quantity and quality. Meanwhile, foreign markets order a number of the triterpene saponin of P. japonicus from China to develop oral iquid, gelatin capsules, and compound recipe tablets, which leads to the fact that the price of P. japonicus constantly goes up.In order to solve the technical bottleneck of industrial development about P. japonicus, we undertake a series of researches. First of all, we focus on the tissue culture and regeneration which satisfy the demand of P. japonicus seedling; secondly, in order to meet the needs of P. japonicus triterpene saponin for medicinal market, the hairy roots culture is continued; thirdly, the cloning of squalene synthase is benefit for supplementing the theory on genetic improvement for high content triterpene saponin; finally, we established the testing methods of total saponin and 5 ginsenosides of P. japonicus roots, at the same time, the dynamic changes of them were analyzed by us. It provided the basic method of inspecting the saponin contents in the course of hairy roots culture and validating the function of SS genes in N.tobacco.. The main results were as follows:1.Bolting the optimal parameter about seed sprouting of P. japonicus by mono-factor and orthogonal experimentsThe results of mono-factor experiment showed that the condition of seed sprouting of P. japonicus is that the optimal GA3 concentration is 150 mg·L-1; seed soaking temperature is 25℃; seed soaking time is 24h; the corresponding germination percentage(GP) is 95%,90%,90% by turns. The results of orthogonal experiment indicated that the optimal seed GP of P. japonicus is A2B2C2, in other words, GA3 concentration is 150 mg·L-1, the required temperature and time is 25℃and 24h. The optimal seed GI of P. japonicus is A2B2C2, that is to say, GA3 concentration is 150 mg·L-1, the temperature is 30℃, the time is 24h.2. Establishing the system of tissue culture and regeneration of P. japonicusOn the ground of MS, the regeneration ratio of adventitious bud is 96.3% in 0.05 mg·L-1NAA+2mg·L-1ZT. It can remarkably increase the quantity of adventitious bud by 0.05 mg·L-1NAA+1.5 mg·L-1ZT, of which incremental coefficient is 9.16. In 1/2MS+2 mg·L-1NAA, the radicated ratio of adventitious bud is 86.4%.3.Establishing on the system of P. japonicus hairy roots culture and analyzing its secondary metabolitesC58C1 can induce P. japonicus to bring about hairy roots. The max inductivity is 90% after tinctured 25 min and 86.9% when co-cultured 4 d. In the course of cultivation, We find it could inhibit the biomass accumulation of P. japonicus hairy roots by using high concentration NAA and IAA; but for IBA, its promotion is only under the condition of 1 mg·L-1 IBA. After the 6-week culture, the biomass accumulation of P. japonicus hairy roots is to the max,increases disobviously along with time-lapse.The reults of HPLC and ultraviolet spectrophotometry showed, in the course of hairy roots cultivation of P. japonicus, it could be divided into three stages:priming stage(1-2 weeks), exponential growth stage(2-8 weeks) and stable equilibrium stage(8-9 weeks). For all monocloning, the dynamic rule of total saponin is PJ9>PJ8>PJ7>PJ6>PJ2>PJ4>PJ1>PJ5>PJ3. Except for PJ3 and PJ5, the synthetical ability of ginsenoside Re was the better, and speed was the most fast and content was the max in all monocloning, Thus, it is clear that ginsenoside Re is the representative matter of P. japonicus hairy roots. Compared with the root of P. japonicus, the 9-week cultured hairy roots'max total saponin of (PJ9), Re (PJ8), Rg1(PJ7), Rg2(PJ3), Rhl (PJ9) and Rh2(PJ6) respectively increased up to 7.5,13.8,28.9,29.9 and 43.8 double.The yields of the essential oils from the roots and hairy roots of p. japonicus are 0.45% and 0.38%, respectively. There are 40 compounds in the essential oils of root samples, and 45 in hairy roots. In the oils of the roots and hairy roots, there were 18 same compounds, their relative peak areas are 59.30% and 59.84%. Among the root oils, the representative matters are hexanoic acid(11.6%), falcarinol(10.04%) and 3-methylbutyric acid(9.56%); and in hairy roots oils, they are caproic acid(13.92%), spathulenol(9.96%) and 1H-cycloprop azulene(9.15%).4. Cloning squalene synthase of p. japonicus and checking its function in N. tobacumBy RACE, we cloned the squalene synthase(SS) of p. japonicus successfully, it is 1353bp, including 45bp 5'-UTR,60bp 3'-UTR and 1248bp ORF. The ORF encodes 415 amino acids, C4054H6790N1320O1706S257 is its molecular formula, the molecular mass is 109559.7 kD; it is hydrophobicital protein. The results of bioinformatics prediction showed that the SS gene is located in cTP, cTP and SP; it owns 2 membrane spaning domains and 8 motif sites. The secondary structure of SS is composed of alpha helix, random coil, extended strand and beta turn.We constructed the plant expression vectors of pCXSN-PjSS and pCXSN-antiPjSS successfully, and transformed the N.tabacum by LBA4404. The detection of PCR manifested that the objective PjSS, antiPjSS and Hyg had integrated into N.tabacum; The amplification of RT-PCR showed that the PjSS in N.tabacum has transcribeed into mRNA, and got its overexpression, but the antiPjSS inhibited its expression. The determination of ginsenoside Re content indicated that the N.tabacum of SS gene could increase its ginsenoside Re content, but the N.tabacum of SS gene was on the opposite.5. Establishing the extraction crafts and content determination of p. japonicus rootThe results of orthogonal experiment indicated that the extraction crafts of p. japonicus root is A2B2C1, that is to said, material/liquid is 1:10, methanol concentration is 65% and temperature is 50℃.The results of ultraviolet spectrophotometry showed that, the colour system was composed of vanillin, perchloric acid, glacial acetic acid and methanol. Under 560 nm, the regression equation of y=0.4024x+0.0043 is constructed by us, R2=0.9999, linearity range is from 0.5μL·mL-1 to 2.5μL·ml-1. The results of reproduction experiment, precision experiment, suitability experiment and recovery experiment suggested that their relative standard deviation (RSD) is less than 5%, and may be used for the content determination of p. japonicus root. The results of HPLC showed, under the 203 nm, the regression equation of ginsenoside Re,Rg1,Rg2,Rh1 and Rh2 were y=42.08x+24.25, y=52.23x+43.87, y=40.09x+3.51,y=115.36x+32.41 and y=98.46x+30.52, their linear range and correlation coefficient are 0.18μg-15.45μg and 0.996～0.998. The reults of reproducibility, stability, precision and recovery test expressed that their relative standard deviation was less than 5% and it is fit for the testing requirement.The reults of contents varying rule suggested, with the growth in year, the content of total saponin increased gradually in p. japonicus root, among which the rangeability of the one-year and the four-year exceeded 50%, and the four-year and five-year was little relatively, only for 9%. The total saponin content of five years p. japonicus root was to the max, for 87.2 mg-g-1. In the different periods of every year, the varying rule of total saponin content was withering period>fruit period>flower period>vegetable growth period. For the same reason, the 5 ginsenosides contents were increasing along the growth year, Five-year root of p.japonicus was to the max. The max content of Re(7.86mg-g-1),Rg1(3.02 mg-g1),Rg2(1.47 mg-g-1) and Rh,(1.72 mg-g-1) was in withering period, and Rh2(1.24 mg-g-1) in fruit period. Compared to four ginsenosides, the Re content was in higher level, and was representative compound of p. japonicus root. |
PDF Full Text Request