| Glycyrrhiza uralensis Fisch. is among the most important and widely used medicinal plants in traditional Chinese medicine. Wild G. uralensis germplasm resources are to be extinguished due to much over excavation and exploitation. Fortunately, artificial cultivation of G uralensis in a large scale of growing areas has been an effective alternative strategy to produce and supply the medicinal plant. However, the glycyrrhizic acid content in cultivated G. uralensis is lower than that in the wild plants. Glycyrrhizic acid is the major chemical to generate medical functions. It is believed that glycyrrhizic acid is mainly responsible for the pharmacological activities of G. uralensis. Therefore, increase of glycyrrhizic acid content in the roots of cultivated plants of G. uralensis would be economically important. The objectives of this investigation were to characterize the effects of applying four micro-elements, B, Mn, Zn, and Mo on the secondary metabolites, primary metabolites, gene of squalene synthase (SQS) andβ-amyrin synthase (β-AS) expression in cultivated G. uralensis. Artificially planted G uralensis was treated with different concentrations of B, Mn, Zn, Mo. The study should provide research-based data for improving cultivation of G. uralensis by increasing its medicinal quality. Major results are described as follows:1. Zn, Mo and Mn had significant effects on quality of G. uralensis although the effects of micro-elements were slight different in foliar application and applying micro-element fertilizers into the soil. Glycyrrhizic acid contents with micro-element spraying treatment were increased at applying Mn and Zn of all three concentrations(0.05%,0.1% and 0.15%), Mo of medium to high concentrations(0.1% and 0.15%).2. Early summer (May) or late fall (October) were the best harvesting months due to higher glycyrrhizic acid contents in roots of G. uralensis. 3. The expression of SQS andβ-AS gene were significantly higher with applying Zn of three concentrations(0.05%,0.1% and 0.15%) and Mo of medium to high concentrations(0.1% and 0.15%) than other treatments.4. Squalene was detected in roots of G. uralensis, but its content was relatively low. Mo with medium to high concentrations(0.1% and 0.15%) and Zn with three concentrations(0.05%,0.1% and 0.15%) elevated the contents of squalene. Squalene content in June was the highest in a year. Significantly positive correlations were found between the contents of glycyrrhizic acid and squalene.5. The chemical composition of the ingredients in the roots of G. uralensis was changed with four micro-elements treatments. Four micro-elements with highest concentration(0.15%) had remarkable influence on crude fiber content in roots of G. uralensis. B, Mn and Zn of medium to high concentrations(0.10% and 0.15%) increaseed the content of crude protein. Mn with three concentrations(0.05%,0.1% and 0.15%) increased crude fat, and so did Mo of medium to high concentrations(0.10% and 0.15%), Zn of low to medium concentrations(0.05% and 0.1%) and high concentration of B(0.15%).6. The relationship between glycyrrhizic acid and other metabolites were analyzed via path correlation analysis. The sequence of direct path coefficient were total flavonoids, polysaccharides, squalene, crude protein, crude fat and liquiritin. The regression equation was: Y=-1.17+0.097x1+0.072x3+1.99x4-0.47x5+0.49x6+0.019x7, in which x1 as crude protein, x3 as crude fat, x4 as squalene, x5 as liquiritin, x6 as total flavonoids, x7 as polysaccharides.7. Relative enrichment in Zn and Mn was found in roots of G. uralensis after B, Mn, Zn, Mo treatments.8. The analyses of Fe, Zn, Cu, Mn, Ca, Mg in G. uralensis showed that the total extraction rate of microelement ranged from 1.71-60.06%, among them the highest was Zn, second was Mn, meanwhile the immerse-residue ratio of Zn was also the highest among the microelements, therefore, Zn could be identified as the characteristic element in G. uralensis. The analyses also indicated that the contents of Cu,Mn,Ca,Mg, and Zn in dissociative and stable forms were more than 50%, the majority of Cu,Mn,Ca,Mg and Zn were in inorganic forms. The second forms of Cu,Fe,Mn,Ca,Mg,Zn in the roots of artificially planted G. uralensis were presented in dissociative and non-dissociative, labile and stable, organic and inorganic forms at the same time. In summary, the glycyrrhizic acid biosynthesis and accumulation were increased by enhancing gene expression of SQS andβ-AS which were two key enzymes in synthesis of glycyrrhizic acid under Zn and Mo treatments, while Mn application enriched its content in the roots and promote the assimilate transportation, consequently enhanced glycyrrhizic acid formation and accumulation.
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