In Vitro Cultures Of Valerian To Biosynthesis Of Valtrate

Valtrate is a pharmacologically important compound. It’s well known thatpharmaceutically active functions of sedative, antineoplastic, increase coronary bloodflow, gastrointestinal smooth muscle, spasm spasmolysis analgesics and antiarrhythmic.Valeriana is difficult to artificial cultivation, and its wild sample has low content ofmedicine ingredients. The cost of traditional valtrate extract method is very expensive.In our research, biosynthesis of valtrate from in vitro culture system was studied bymethods of Cell Culture, GC-MS, HPLC-PDA, Plackett–Burman Design, Box–Behnken Design and RT-PCR. Therefore, we focused on analysis of the main chemicalconstituents of3kinds of Valeriana, establishment of valtrate high yield culture system,optimization of culture condition to enhance valtrate accumulation establishment ofkinetic model of adventitious roots suspension culture system, optimization ofregulation factors to enhance valtrate accumulation and analysis of the effect ofregulation factors on expression of dxr gene. The results are showed as follows:Through GC-MS analysis,43kinds of chemicals were identified from the essentialoil of Valeriana amurensis Smir. ex Kom., the maximum content of Isobornyl acetatewas27.6%, which was the highest value in reported research.44kinds of chemicalswere identified from Valeriana fauriei Briq. essential oil, and α-Selinene (16.7%) wasthe main constituent.39kinds of chemical were identified from Valeriana alternifoliaBunge. essential oil, and Bornyl acetate (39.1%) was the main constituent.4kinds ofvalepotriates (Valtrate, Isovaltrate, Diavaltrate and Acevaltrate) were identified byHPLC. Valtrate was the main constituent, which has best medicinal properties.Through screening of explants, culture medium and hormone combination, callusand adventitious roots were induced. Then, callus, suspended cell, solid and suspendedadventitious roots culture system were established. The highest content of valtrate (1.77times than wild root sample) was gotten from suspended adventitious roots culturesystem which was chosen as the high-yield cultlure system. Plackett–Burman designcriterion was applied to identify the significant effects of various cultural parameters.Among the various variables screened, the rotating speed, NH4NO3and KH2PO4concentrations were most significant factors. Through the Box–Behnken design, thespecific optimum levels of these significant parameters were determined as follows: the rotate speed (76r/min), NH4NO3concentration (0.0069mol/L) and KH2PO4concentration (0.0436mol/L). And the maximum valtrate content (8.46mg/g,6.79times than wild root sample) was gotten under this optimized conditions. Using theOrigin software to non-linear fit the experimental data, the kinetics model of tissuegrowth, valtrate production and substrate consumption were established. Thismathematical model can interpret and reflect the change of key parameters in theprocess of suspension adventitious root culture.We assayed the effects of different regulation factors on the production of valtrate.Methyl jasmonate, Jasmonic acid and Chitosan showed the significant promotion effecton valtrate production. The test results of precursor feeding indicated that citronellal hada modest promotion effect on valtrate production. Using the response surfacemethodology, the optimal combination of methyl jasmonate, jasmonic acid and chitosanwas shown to have strong synergistic effect on valtrate production, which wasconfirmed by actual experiments. After the test of best time of induction treatment, wegot the maximum valtrate content of17.40±0.26mg/g, which was14.15times thanwild root sample, and this was the highest value in reported research.DXR is a key enzyme in terpenoid metabolic pathways. One dxr gene was clonedfrom cell of V. amurensis by RTPCR. The cDNA fragment code region of dxr was1425bp long, encoding474amino acids. According to the test results of prokaryoticexpression, a fragment about50.98kD was achieved as expect. This order of geneexpressed strength from different tissues of V. amurensis is: adventitious roots＞wildroot&rhizome＞wild leaf＞suspended cell. The effect of regulatory factor on theexpression of dxr gene has positive correlation with the effect of regulatory factor onproduction of valtrate. The results show dxr gene is an effective gene target in thesynthesis pathway of valtrate. Through the research of establishment of valtrate highyield culture system, the culture condition was optimized, the kinetic model ofadventitious roots suspension culture system was established, the regulation factors wasoptimized, and the valtrate production level was significantly enhanced. Further studieshave shown that the biosynthesis regulatory factors can not only promote the synthesisof the target compound, but also enhance the level of dxr gene expression. The results ofthis study provide the theoretical basis for large-scale biosynthesis of varltrate, and arehighly significant to reveal the mechanism of varltrate biosynthesis.