Molecular Markers Of High Glycyrrhizic Acid Content Based On SNPs Of Isopentenyl Transferase Gene

Glycyrrhizae Radix et Rhizoma, known to be the ’king’ of traditional Chinese medicine, is the most commonly used medicinal material and important additive, which can be medicine as well as food. Glycyrrhizic acid, a triterpenoid saponins component, is the main active component in the root and rhizome of licorice. However, the content of glycyrrhizic acid in cultivated licorice, which has become the mainstream because of lacking wild resources, is generally low and is difficult to reach the standard of Chinese pharmacopoeia. Moreover, individual difference of glycyrrhizic acid content is obvious. The above problems will seriously restrict the clinic use and preparations production of licorice. Therefore, research on the reasons of cultivated licorice quality declining and how to improve it to maximumly close to wild licorice is the key problem to solve.In the previous studies of our laboratory, they have found polymorphism of functional genes HMGR、SQS、β-AS, which are in the glycyrrhizic acid biosynthesis pathway, could explain part genetic variation mechanism of glycyrrhizic acid content. In addition, we have considered that there is an interrelated and interacted metabolic network consisted of secondary metabolites biosynthetic pathways in the plant through reviewing a large number of papers. In this paper, we tried to determine content of glycyrrhizic acid and cytokinins after the exogenous cytokinins stimulation and in the natural condition combining HPLC and ELISA techniques, and analyze the correlation between glycyrrhizic acid content as well as cytokinins content. Then we attempted to clone the full-length of IPT gene, the key enzyme gene in the synthesis of cytokinins, and select the SNPs of IPT gene via PCR-sequencing, based on biosynthetic pathway of cytokinins which is an important phytohormone and has cross node with glycyrrhizic acid biosynthetic pathway. Finally, we intended to obtain the molecular marker of high glycyrrhizic acid content based on the SNPs of IPT gene by using statistical software. The purpose is to reveal molecular mechanism of glycyrrhizic acid genetic variation, to provide a significant basis for improving the glycyrrhizic acid content in cultivated licorice and breeding good varieties.The main results are as follows:(1) The glycyrrhizic acid content was analyzed after exogenous cytokinins (6-BA) stimulating. The leaves of licorice plant were sprayed with exogenous cytokinins 6-BA, then dynamic change of glycyrrhizic acid content was determined via HPLC. The result showed that glycyrrhizic acid contents of the majority 6-BA processed licorice were obviously higher than the control in each sampling period (P<0.05). The most significant effect was 100 mg·L-1 6-BA processing in June, and the increase rates of five sampling were 38.02%,72.31%, 7.52%,59.34%,43.30%, respectively. What’s more, 15mg·L-1 in June as well as 25mg·L-1, 100mg·L-16-BA processing in July could also obviously accelerate the accumulation of glycyrrhizic acid.(2) The content of glycyrrhizic acid and cytokinins in the licorice were analyzed. The glycyrrhizic acid and cytokinins (ZR,DHZR,IPA) contents in 98 licorice, which had the same growth environment and age, were determined by HPLC and ELISA techniques. The result indicated that individual difference of glycyrrhizic acid content is obvious, and the variation range was 0.61%～3.87%. The main cytokinins content was relatively low and had little difference in sampling period. The statistical analysis result indicated that the contents of cytokinin and glycyrrhizic acid exist obviously positive correlation, verifying that glycyrrhizic acid content was improved after exogenous cytokinins stimulating.(3) The full-length sequence of licorice IPT gene was obtained. The length was 1002 bp, which included just 1 extron, coding a 333 amino acid residues protein.(4) The SNPs of licorice IPT gene were selected by PCR-sequencing. Five SNPs were found through determining the above 98 licorice, including 161bp G/A,217bp G/A,348bp T/A,821bp A/G,848bp T/A. Among them,348bp existed missense mutation and the rest SNPs were synonymous mutations. According to the five SNPs, the licorice samples could be divided into four genotypes respectively, including Gl:G-G-T-A-T, G2:G-G-A-A-T, G3: A-A-A-G-A, G4:G/A-G/A-T/A-A/G-T/A. The former three were homozygous type and the last was hybrid type.(5) The correlation between SNPs of licorice IPT gene and high glycyrrhizic acid content was analyzed. The glycyrrhizic acid contents of four IPT genotypes had obvious difference (P<0.05); Among five SNPs,348 bp was the main associated SNP, while 821 bp and 848 bp were auxiliary associated SNP, and cytokinins as well as glycyrrhizic acid content might be the combined action of one or more SNPs. Combining SNPs of liquorice IPT gene, cytokinin and glycyrrhizic acid content, the result preliminarily showed the content of glycyrrhizic acid was 348bp T> A,821bp A>G,848bp T>A, and hybridization was probably between the two bases. In conclusion, licorice IPT gene SNPs could be a molecular marker of high glycyrrhizic acid content.