| Many studies have shown the activity of some enzymes can be enhanced by adjusting environmental factors, such as ultraviolet light intensity, temperature, humidity, which consequently improves the concentration of specific active ingredients of the medicinal plants significantly and produces new highly active compounds. This experiment technology is defined as active-enzymes induction technology. The experimental subject of this topic is the leaves of Catharanthus roseus(CR). By applying active-enzymes induction technology, the internal content of terpenoids indole alkaloids can be increased. Supplemented with RT-qPCR technology, the difference of gene expression of key enzymes responding to the active-enzymes induction have been found, and this can explain the impact of active-enzymes induction on improving the concentration of specific active ingredients of the medicinal plants in gene level.1. The postharvest CR leaves were induced by active-enzymes induction. By adjusting the ultraviolet light intensity, the induction time, training time and other factors, the optimum induction conditions (the ultraviolet light intensity was120.83μw·cm-2, the induction time was1h, the darkroom incubation time was72h, the darkroom incubation temperature and humidity were respectively25℃and80%) were well-defined. After inducing, content of following four compounds:strictosidine, vindoline. catharanthine and ajmalicine changed dramatically, the content of strictosidine and ajmalicine respectively increased by76.4%and405.8%,the content of vindoline and catharanthine respectively decreased by21.3%and27.5%.2. The CR plants were induced under the same condition, the content of strictosidine, vindoline, catharanthine and ajmalicine in these leaves significantly changed. The content of strictosidine and ajmalicine respectively increased by527.9% and323.6%, the content of vindoline and catharanthine also increased20.1%and19.0%.3. By using RT-qPCR technology, the influence of the content of secondary metabolites in CR after active-enzymes induction can be interpreted from the gene level. Considering the instantaneous expression characteristics of RNA, the CR plant induced by active-enzymes induction with darkroom incubation. Selecting four inducing time(0min,5min,30min and60min), the difference of relative expression of gene TDC, G10H,SLS, STR, SGD, T16H, D4H, DAT and ORCA3were investigated. Expect gene STR, T16H and D4H, whose inducing time of the maximum relative expression were15min, all the other inducing time of the maximum relative expression were30min. The relative expression of gene TDC, SLS and SGD was down-regulated after induced60min. The relative expression of transcription activator ORCA3was up-regulated significantly. The relative expression of gene TDC, STR and SGD which under the control of ORCA3also up-regulated significantly.4. The physiological activity of CR which induced by the best induction conditions showed that the photosynthetic pigment did not significantly change, the content of MDA, proline, soluble sugar and soluble protein significantly decreased, expect the content of GSH, the activity of antioxidant enzymes significantly increased, except the activity of GR. These multiple systems worked together to reduce the physiological influence on CR after the active-enzymes induction.In this study, the fact of active-enzymes induction technology enhancing the content of strictosidine and ajmalicine significantly, provides a cogent evidence of that this technology can optimize the quality of medicinal herbs and enrich herb resources. By applying RT-qPCR technology, the key genes of key enzymes in terpenoids indole alkaloids synthesis pathways of CR responded to the active-enzymes induction were found; this lays a good foundation for interpreting the active-enzymes induction mechanism from gene level,The expression of these genes can be controlled, which can promote to establish a new methodology of directed improvement of content of secondary metabolites in plant. |
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