Molecular Mechanism Of Biosynthesis Of Licorice Flavonoids Based On Polymorphism Of CHS Gene

The root and rhizome of licorice is one of the most frequently-used Chinese herbs with the functions of nourishing qi,alleviating pain,tonifying spleen and stomach,eliminating phlegm,and relieving coughing.Chinese Pharmacopoeia clearly stipulates that the content of liquiritin(C₂₁H₂₂O₉)in licorice samples should not be less than 0.50%.However,based on our previous investigation and analysis,the content of liquiritin in most of licorice cultivar samples is hard to meet the minimum requirement in Chinese pharmacopoeia,presently.Chalcone synthase?CHS?is a key and rate-limiting enzyme involved in the biosynthetic pathway of liquiritin,and it plays an important role in regulating the biosynthesis of flavonoids in licorice.Therefore,exploring the polymorphism of CHS and its mechanism in flavonoids accumulation from licorice has an important theoretical value and practical significance for improving the quality of cultivated licorice and guiding the licorice molecular breeding.In this paper,the content of liquiritin,isoliquiritin,liquiritigenin,and isoliquiritigenin in 60 cultivated licorice samples were assayed by HPLC.The cDNA sequences of CHS were amplified by RT-PCR method from roots of licorice and analyzed by DNAMAN.Then the relationship between the content of flavonoids and the single nucleotide polymorphism?SNP?of CHS was analyzed,and the specific CHS genotypes corresponding to the high/low content of flavonoids were determined.The recombinant expression vectors,pPIC9K-CHS,were constructed and transformed into disarmed Pichia pastoris GS115 by electroporation.The expression of CHS was induced by methanol and detected by SDS-PAGE.The results of this paper are as follows:?1?HPLC results:HPLC was used to assay the content of liquiritin,isoliquiritin,liquiritigenin,and isoliquiritigenin in 60 licorice root samples from three origins.The results showed that the contents of these four main flavonoids were significantly correlated in all samples.And there was an obvious difference in the contents of the four flavonoids among three different original licorice samples.The contents of the four flavonoids were highest in Glycyrrhiza uralensis samples,lowest in Glycyrrhiza inflate samples,and centered in Glycyrrhiza glabra samples.According to the above results,group 1?five G.uralensis samples with the highest contents of flavonoids?and group 2?five G.inflate samples with the lowest contents of flavonoids?were determined for further cloning and analysis of the CHS cDNA sequences.?2?CHS cDNA cloning results:336 CHS cDNA sequences with a full length of 1175 bp encoding 389 amino acids were cloned.These 336 CHS cDNA sequences with 249 variable sites were divided into 137 haplotypes?haplotype 1-137?,and the similarity of which was 98.98%.130 variable sites were present in the 336 amino acid sequences,and 102 types were determined?AA1-102?,the similarity of which was 99.40%.All sequences have been registered in GenBank Database.?3?CHS polymorphism analysis results:By analyzing the 137 CHS genotypes and the 102 CHS amino acid sequences types,we found that the main CHS genotypes in all licorice samples were haplotype 10 and 92 which encoded AA-3 and AA-60,respectively.The main CHS genotypes corresponding to the high/low contents of flavonoids were haplotype 60 and 63 which encoded AA-35 and AA-36,respectively.?4?Bioinformatics analysis results:A significant correlation was present between the mutations at 193rd and 229th amino acid residues of AA-35 and AA-36 and the accumulation of flavonoids by bioinformatic analysis.Physicochemical properties analysis results:The molecular size of CHS from licorice was about 43 KDa,and there was no significant difference between AA-35 and AA-36 in isoelectric point,unstable coefficient,and hydrophilic coefficient.They also had the same half-life periods.Secondary structure analysis results:The random coil/extension difference was present at the 193rd and 225th amino acid residues and the ?-spiral/extension difference was present at the 237th amino acid residue of AA-35 and AA-36.These differences between the secondary structures of AA-35 and AA-36 were consistent with the differences between the primary structures of AA-35 and AA-36.Both of them had the same active site?the 164th amino acid residue?and active region?from the 156th to the 172nd amino acid residues?.The tertiary structure analysis results:The homology modeling results of AA-35 and AA-36 showed that the homology structures had good stereochemical parameters and spatial structure,and could be used for further analysis.The binding sites analysis showed that only valine at the 229th amino acid residue of AA-35 was in the binding site regions,which could bind to malonyl-CoA,while isoleucine at the193rd amino acid residue of AA-35,valine and threonine at the 193rd and 229th amino acid residues of AA-36 fell flat with the substrate binding.It explained the correlation between mutations at the 193rd and 229th amino acid residues of AA-35 and AA-36 and the high/low levels of flavonoids from the angle of molecular level.Cluster analysis results:Through the construction of phylogenetic tree,it was found that the amino acid sequences of CHS had a good discrimination between different species,and the results were consistent with the evolutionary relationship.The main CHS amino acid sequences in licorice,AA-3 and AA-60,were clustered together first,then they were clustered together with AA-36,and finally with AA-35.It explained the obvious difference between AA-35 and AA-36 from the angle of evolutionary relationship.?5?Heterologous expression results:The recombinant Pichia pastoris GS115-P-CHS10,GS115-P-CHS92,GS115-P-CHS60,and GS115-P-CHS63 which contained specific CHS genotypes were successfully constructed.PCR and sequencing validations were performed with the above recombinant P.pastoris after the His⁺ transformant selection,geneticin G418 selection and Mut+ recombinants selection.Methanol was used to induce the expression of those specific CHS genotypes in GS115-P-CHS10,GS115-P-CHS92,GS115-P-CHS60,and GS115-P-CHS63,and SDS-PAGE was used to detect the results,which showed a 43 kDa protein was obtained with the same size of CHS.