Molecular Markers Of High Glycyrrhizic Acid Content Based On SNPs Of Bacillus Lycopene Synthase Gene

Liquorice, consisting of the roots, rhizomes, and stolons of Glycyrrhiza uralensis Fisch. or G. inflata Bat. or G. glabra L., is widely distributed in the middle-east, mediterranean area and north of China. The main bioactive constituents of licorice are triterpene saponins and flavonoids, with analgesic, antitussive, anti-inflammation, antitumor and other pharmacological activities. Nowadays, the cultivated G. uralensis is increasingly becoming the main commodity in pharmaceutical market because of the longstanding over-harvesting of wild liquorice and environmental deterioration in China. But how to cultivate the high quality G. uralensis is an urgent issue to be settled. So, clarifying the regulatory network among chemical ingredients and finding the chemical labeling may help augment the main medicinal ingredients in G. uralensis.The presented research is aimed to explore the effect of exogenous abscisic acid (ABA) stress on chemical constituent network of G. uralensis. The regulatory network of G. uralensis was investigated via detecting the content of seven main medicinal ingredients with abscisic acid stimulating. The transplants of two-year-old G. uralensis were subjected to four concentration of ABA (25,50,100,200 mg/L) in three different growth periods (June, July and August). Subsequently, the samples were collected every month after the treatment. Then the long-term dynamic changes of seven kinds of chemical composition (glycyrrhizic acid, liquiritin, isoliquiritin, liquiritigenin, isoliquiritigenin, liquiritin apioside, liquiritin isoapioside) were analyzed by high-performance liquid chromatography (HPLC). Then the relationship between endogenous ABA and glycyrrhizic acid was analyzed to obtain the chemical labeling with high glycyrrhizic acid content.The main reason for the difference in glycyrrhizic acid content was genetic factors. Previous studies on the genetic mechanism of the changes in the glycyrrhizic acid content usually focused on the functional genes of the biosynthesis of the acid, which can’t explain all the changes. In this study, we reveal the genetic mechanism by studying the SNPs with SPY gene, which was a key enzyme gene in the synthesis of ABA.The results of this paper are as follows:(1) The content of these composition rose remarkably after ABA stimulating in June and July. The content of glycyrrhizic acid, liquiritin, isoliquiritin, isoliquiritigenin and liquiritin isoapioside increased remarkably after one month under 25mg/L ABA stimulating in June, the increase rates were:79%、26%、20%、19% and 18%, respectively. The content of liquiritigenin and isoliquiritigenin increased remarkably after four months under 50mg/L ABA stimulating, the increase rates were:24%、 79%, respectively.(2)The content of glycyrrhizic acid, liquiritin, isoliquiritin, isoliquiritigenin and liquiritin isoapioside increased remarkably at September 20th sampling after 50mg/L ABA stimulating, the increase rates were:78%,131%,166%,113% and 97%, respectively. The content of liquiritigenin and isoliquiritigenin increased remarkably at August 20th sampling after 50mg/L ABA stimulating, the increase rates were:66%、 148%, respectively.(3) It is revealed that ABA stress prevented the decrease of flavonoids in G. uralensis under natural growth conditions. And the treatment of ABA had the on the content of chemical constituents of G. uralensis, but hardly altered the ratio among their chemical composition.(4) Under the condition of natural cultivation, there were significant differences between the content of ABA and glycyrrhizic acid in the different habitats (P<0.05), which showed that the most probable cause of this difference was genetic factors.(5) The full-length sequence of PSY gene was obtained. The gene which included 5 extrons and 4 introns was 2770 bp, the ORF was 1206, coding a 401 amino acid residues protein.(6) PSY genes were obtained from 59 samples with 12 regions by RT-RCR-cloning technique. After analyzing the sequences of the exon,5 SNP loci were obtained, which were in 118bp,273bp,1095bp,1104bp,2660bp, respectively. Using MATLAB7.0 data processing software, the correlation degree between the 5 sites and the glycyrrhizic acid was sequenced as 2660bp> 273bp> 1104bp> 118bp> 1095bp.(7) According to the provisions of the 2015 edition of Pharmacopoeia, the annual licorice with high glycyrrhizia acid content was dividen into high glycyrrhizia acid group, the other into low glycyrrhizic acid group. After the classification of 59 samples from different habitats, the genes were divided into 6 types, which were P1(118C-273A-1095G-1104C-2660A), P2 (118C-273A-1095A-1104C-2660A), P3 (118C-273A-1095G-1104C-2660G), P4 (118C-273G-1095A-1104C-2660A)、P5 (118T-273A-1095A-1104C-2660A), P6 (118C-273A-1095A-1104G-2660A), respectively. Type P2 was the largest haplotype, accounting for 83% of the whole samples. All samples with type P1 were in high glycyrrhizic acid group, and samples with the other genotype were in low glycyrrhizic acid group. Type P1 might be used as a molecular marker for selecting high quality licorice.