Rapid Accumulation Mechanism Of Gene-based SNPs β-AS Genuineness Of Licorice And Licorice Acid

Glycyrrhiza uralensis is believe to be one of the most important herbs in traditional Chinese medicine for its numerous functions. The content of glycyrrhizic acid, which is the most important therapeutic compound of the herb, is an important indicator for the evaluation of the the quality of licorice. However, the content of glycyrrhizic acid in cultivated licorice is lower than the wild licorice, which will influence the clinic efficacy of licorice and quality of traditional Chinese medicine. So the more the quality of cultivated licorice closes to wild licorice, the better the quality of cultivated licorice is. Revealing the genuineness of licorice is the key to solve this problem.In our previous studies, we have found there are two SNPs in the94th bp and254th bp of beta-amyrin synthase (β-AS) gene. In the254th bp, the C turns into a T. In the94th bp, the G turns into an A which leads to the amino acids this part coded turns from Glycine to Aspartate. What’s more, in these variations, the G94A and C254T genotypes have a significan correlation with high content of glycyrrhizic acid. In this paper, we tried to reveal the genuineness of licorice by cloning the full-length sequence of β-AS gene and analyzing the correlations among SNPs with regions, content of glycyrrhizic acid and rapid accumulation of glycyrrhizic acid.In this paper, PCR and sequencing was used to clone the full-length of β-AS gene, and PCR-SSCP (Single-Strand Conformation Polymorphism) was used to select the SNPs of β-AS gene. To reveal the genuineness of licorice, the content of glycyrrhizic acid we had obtained was used to analyze the correlations between SNPs and regions.The results of this paper are as follows:1.The full-length sequence of β-AS gene was obtained. The gene which included15extrons and14introns was4109bp, coding a762amino acid residues protein.2.The SNPs of β-AS gene was selected by PCR-SSCP. After selecting102licorice from7regions, a SNPs in the94th bp of β-AS gene was found. According to the SNPs, the licorice could be divided into3genotypes, respectively94A type,94G type and94A/G type. Two SNPs in the1st intron of β-AS gene was found after selecting. According to the SNPs, the licorice could be divided into4genotypes, respectively506C-512C type,506C/G-512C/G type,506C-512C/G type and506C/G-512C type. According to the3SNPs in the extrons and introns, the licorice could be divided into7genotypes, respectively94A/G-506C-512C type,94G-506C-512C type,94A/G-506C/G-512C type,94A-506C-512type,94A/G-506C/G-512C/G type,94G-506C-512C/G type and94A/G-506C-512C/G type.3.The correlations between SNPs of β-AS and genuineness were analyzed. The percentage of94A type in genuine regions was31.7%, the percentage in non-genuine regions was5.9%, there were significant differences between them (P=0.000). There were no significant differences between the percentages of94G type and94AG type in genuine regions and non-genuine regions. The percentage of506C-512C type in genuine regions was78.6%, the percentage in non-genuine regions was55.6%, there were significant differences between them (P=0.014). There were no significant differences between the percentages of3other types in genuine regions and non-genuine regions.Combining the SNPs in the extrons and introns, the licorice could be divided into7genotypes. There were4genotypes in both genuine regions and non-genuine regions. In these genotypes, the percentage of94A-506C-512C type in genuine regions was31.7%, the percentage in non-genuine regions was5.9%, there were significant differences between them (P=0.000). There were no significant differences between the percentage of94A/G-506C-512C type,94G-506C-512C type and94A/G-506C/G-512C type in genuine regions and non-genuine regions.4. The correlations between SNPs of β-AS and rapid accumulation of glycyrrhizic acid were analyzed. According to Chinese Pharmacopoeia, the content of glycyrrhizic acid in licorice must be more than2.0%. We divided the annual licorice of which the content of glycyrrhizic acid were more than2.0%into rapid accumulation group, the annual licorice of which the content of glycyrrhizic acid were less than2.0%into control group. In the4genotypes divided by the SNPs in intron of β-AS, The percentage of506C/G-512C type in rapid accumulation group was45.5%, the percentage in control group was13.0%, there were significant differences between them(P=0.014). There were no significant differences between the percentages of3other genotypes in rapid accumulation group and control group. In the7genotypes divided by the SNPs of P-AS, there were4genotypes in both rapid accumulation group and control group. The percentage of94A/G-506C-512C type in rapid accumulation group was18.2%, the percentage in control group was52.2%, there were significant differences between them (P=0.042). There were no significant differences between the percentages of94A-506C-512C type.94G-506C-512C type and94A/G-506C/G-512C type in rapid accumulation group and control group. There were no significant differences between the percentages of94A type,94G type and94A/G typed in rapid accumulation group and control group (P=0.809、P=0.245、P=0.270).506C/G-512C type might be used as a detection index to select high quality licorice, while type might be used as a decetion index to screen poor quality licorice.