| Radix glycyrrhizae is a commonly-used bulk medicinal material and has been called the National Venerable Master. It has been applied in a large amount both at home and abroad. Yet, the wild radix glycyrrhizae resource is becoming deficient, which has driven the cultivated radix glycyrrhizae to become a mainstream product. Most of the cultivated radix glycyrrhizae, however, has poorer quality. Its glycyrrhizic acid, for example, fails to reach the ratio of 2.0% and 2.5% as required in Pharmacopoeia of China (version 15) and Pharmacopeia of Japan (version 16), respectively. If this problem cannot be solved, radix glycyrrhizae will have no guaranteed clinical curative effect and its import will be restricted. In this case, improving quality of cultivated radix glycyrrhizae has become an urgent problem to solve. To solve that problem, our research team conducted substantive research and looked up many literatures, discovering that the synthetic pathways of secondary metabolic products in plants have formed an interrelated and interactional metabolism network. Inspired by this finding, this paper started from the regulation network promoting glycyrrhizic acid synthesis, screened out endogenous regulation substances for promoting glycyrrhizic acid synthesis, cloned, analyzed and functionally verified key genes of such substances and performed induction factor treatment, to analyze the mechanism of promoting glycyrrhizic acid synthesis of such substances, both molecularly and chemically. Variation in the content of regulation substances in different germplasms has been studied, to learn about the content of the substance in the natural state of different germplasms. The molecular mechanism accounting for such variation has been analyzed based on the relevance between polymorphism of key functional genes regulating synthesis of such substance and the content of the regulating substance, with the aim to provide basis for screening radix glycyrrhizae and other medicinal plants of good quality. Results are as follows:1. Multiple hormones of different concentrations were applied externally to screen regulation substances promoting glycyrrhizic acid synthesis. During the middle ten days of June (preliminary growth stage) and July (middle growth stage), ABA, GA, MeJa,6-BA, BR and IAA of different concentrations were sprayed on radix glycyrrhizae. HPLC method was used to measure the content of glycyrrhizic acid, glycyrrizin and five flavonoids (isoliquiritin, liquiritin apioside, isoliquiritin apioside, glycyrrhizin and isoliquiritigenin) in July, August, September and October (four sampling periods), respectively. Results have shown that:firstly, the treatment concentration 25mg/L of ABA applied in the middle ten days of June had better promotion effect on glycyrrhizic acid and glycyrrizin, with the glycyrrhizic acid content 78.96%,52.28%,46.43% and 52.01% higher and the glycyrrizin content 25.61%,47.80%, 101.62% and 27.85% higher than the control group, in the four sampling periods, all having reached the significance or the extreme significance level. The treatment concentration 50mg/L of ABA applied in the middle tends of July had better promotion effect on glycyrrhizic acid, but all concentrations produced weaker promotion effect on glycyrrizin that that in June. The influence from ABA of different concentrations on five flavonoids of radix glycyrrhizae was not significant. Secondly, the treatment concentration 100mg/L of 6-BA applied in the middle ten days of June had better promotion effect on glycyrrhizic acid and glycyrrizin, they were significantly higher than the control group in the four sampling periods in July. The treatment concentration 100mg/L of 6-BA applied in the middle ten days of July also had better promotion effect on glycyrrhizic acid and glycyrrizin, the glycyrrhizic acid content were remarkable higher than the control group in the three sampling periods. The influence from 6-BA of different concentrations on five flavonoids of radix glycyrrhizae was not significant. Thirdly, the influence from GA, MeJa, BR and IAA on glycyrrizin and glycyrrhizic acid of glycyrrizin was weaker than ABA and 60BA, with insignificant difference from the control group. Besides, their influence on other five flavonoids was instable, with insignificant difference on the whole. Fourthly, among the six hormones, ABA produced the best treatment effect, with 60BA coming next. The two hormones cannot only significantly improve the glycyrrhizic acid and glycyrrizin content in glycyrrhizic acid, but also have smaller influence on other five flavonoids. GA could promote glycyrrhizic acid and glycyrrizin, but influence other five flavonoids in an instable manner. MeJa was the worst player in promoting glycyrrhizic acid and glycyrrizin. Fifthly, low-concentration ABA can promote seven components of radix glycyrrhizae, but the high-concentration ABA works contrarily. ABA with concentration ranging 0-50mg/L had extremely significantly positive correlation with glycyrrizin, isoliquiritin and glycyrrhizic acid (P<0.01) and significantly positive correlation (P<0.05) with isoliquiritin apioside. Whereas, ABA with concentration ranging 50-200mg/L had negative correlation with all seven components and extremely significant negative correlation (P<0.01) with glycyrrhizic acid. The correlation between other five hormones and seven components of radix glycyrrhizae was shown differently. We can see that ABA is a regulation substance to better promote glycyrrhizic acid synthesis.2. ABA’s mechanism of promoting glycyrrhizic acid synthesis was studied. Taking radix glycyrrhizae samples applied with ABA of different concentrations in the middle ten days of June (preliminary growth stage) and July (middle growth stage), growth features and output indicators of radix glycyrrhizae plant were measured. RNA extraction and reverse transcription response were conducted taking radix glycyrrhizae seedlings applied with ABA of different concentration as materials. Results have shown that:firstly, ABA of different concentration had influence of different degrees on the growth indicators of the biennial radix glycyrrhizae plant at the preliminary treatment period. During the first sampling in the two treatment periods, samples applied with 100mg/L and 200mg/L ABA had lower plant height, ground diameter, root width and root fresh weight than the control group; however, this inhibition gradually disappeared in company with the growth of radix glycyrrhizae. By contrast, the 25mg/L and 50mg/L ABA had insignificant influence on growth of radix glycyrrhizae on the whole. Secondly, ABA of different concentrations could promote β-AS expression in a short period, while high-concentration ABA would inhibit the expression if applied for a long period. When radix glycyrrhizae seedlings were treated with ABA of different concentrations, at 3h and 6h, all treatment could promote β-AS expression, showing significant difference from the control group. Yet at 12h and 24h, samples applied with 100mg/L and 200mg/L ABA had significantly lowered β-AS expression and at 24h, the expression of both concentrations was significantly lower than the control group.3. NCEDs gene family members of radix glycyrrhizae were cloned and processed with bioinformatics analysis. Taking the entire radix glycyrrhizae seedling as material, total RNA was extracted and reverse transcription was performed. The NCEDs gene family members (NCED1, NCED3, NCED4) were cloned. Results have shown that:the open reading frame length of NCED1, NCED3 and NCED4 was 1830bp,1830bp and 1764bp, respectively. All of the three genes had one exon, but no intron. They could be coded with 609,609 and 587 amino acids. The molecular weight of their hypothetical protein was 67.56 kDa,67.29kDa and 63.98kDa, respectively, and their isoelectric point 6.64,5.95 and 6.26, respectively.4. Effective ingredients like glycyrrhizic acid and variation in hormones in radix glycyrrhizae of different origins were studied. Taking roots of radix glycyrrhizae that had been sampled as material, the principal root was used for measuring the content of glycyrrhizic acid, glycyrrizin, isoliquiritin, glycyrrhizin and isoliquiritigenin, and the root tip for measuring the content of endogenous 6-BA, ABA, GA, MeJa and BR. Results have shown that:firstly, the difference in glycyrrhizic acid content between different origins reached the significance level (P<0.05). Among the 13 origins, Yingxian County (Shanxi Province) enjoyed the highest glycyrrhizic acid content, and Minqin (Gansu Province) had the lowest content 1.28%. Among different origins, Bayinwusu Town, Hangjin Banner, Inner Mongolia had the greatest extreme difference in the glycyrrhizic acid content, with the difference of 2.95%; and the least difference 0.98% was found in Aohan Banner, Inner Mongolia. The ratio between the greatest and the least difference was 33.22%. The difference in glycyrrizin, isoliquiritin and isoliquiritigenin among different origins reached the extreme significance or significance level on the whole. The glycyrrhizin content among different origins on the whole saw insignificant difference. Secondly, difference in ABA content from different origins reached the extreme significance level (P=0.002<0.01). Eight out of the 13 origins had the ABA extreme difference ranging 40ng·g-1.FW-100ng·g-1.FW, one less than 40ng·g-1.FW and four more than 100ng·g-1.FW. Thirdly, difference in 6-BA, MeJa and GA content from different origins reached the extreme significance, significance and extreme significance level, respectively, but the difference in BR did not reached the significance.5. There were 65,67 and 36 samples were successfully cloned and sequenced in the 13 provenances samples for the NCED1, NCED3 and NCED4 gene, and 14,14 and 29 SNPs were found,respectively.22 genetypes were found according to the NCED1 gene,29 genetypes were found according to the NCED3 gene and 27 genetypes were found according to the NCED4 gene. There were many genetypes in one provenance for the 3 genes and party provenances had different genetypes, it indicated that there are rich variation in the internal provenance. And the genetype for the samples form different provenances was same, it indicated that there are consistency in the different provenances.6. Molecular mechanism in glycyrrhizic acid and ABA content variation was studied. Experiment data in Chapter Six and Seven were used for grey correlation analysis between the radix glycyrrhizae NCEDs gene SNPs loca and the ABA & glycyrrhizic acid content. Results have shown that:firstly, the variation loca of all SNPs of NCED1 gene, NCED3 gene and NCED4 gene having the correlation coefficient with the glycyrrhizic acid content greater than 0.8 took up 78.57%,76.92% and 72.41% of the total, respectively, indicating that the three genes had strong promotion effect on synthesis and accumulation of glycyrrhizic acid. Secondly, the variation loca of NCED1 having the correlation coefficient with ABA content greater than 0.8 took up 78.57% of the total. However, all variation loca of NCED3 had the correlation coefficient ranging 0.8-0.7. All variation loca of NCED 4 having the correlation coefficient greater than 0.7 took up 68.97% of the total. This indicates that among the three genes, genovariation loca of NCED1 had better performance in ABA synthesis and accumulation than the other two genes. The other two genes also had promotion effect on ABA synthesis and accumulation.In conclusion, the genetypes of A type for NCED1 in 437bp, A type for NCED3 in 966bp, G type for NCED1 in 845bp should be considered when take the selective breeding for the Glycyrrhiza uralensis Fisch. |
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