Research On The Molecular Mechanism Of Glycyrrhiza Flavonoids Biosynthesis Based On CHI Gene Polymorphism

Licorice is one of the most-frequently-used Chinese herbs,which was first recorded on Sheng nong ben cao jing.Because of the significant functions of licorice,nourishing qi,alleviating pain,tonifying spleen and stomach,eliminating phlegm,and relieving coughing,there is a large market demand of licorice.Chinese Pharmacopoeia clearly stipulates that the content of liquiritin(C21H22O9)in licorice slices should be no less than 0.50%.However,there are lots of unqualified licorice slices in Chinese herbal medicine markets,which directly influence the pesticide effect of licorice.Chalcone isomerase(CHI)is the second-limiting and key enzyme involved in the biosynthetic of liquiritin,which regulates and controls the accumulation of liquiritin.Thus,this study mainly concentrated on the polymorphism of CHI,revealing the molecular mechanism of the licorice flavonoids biosynthesis,and guiding the breeding of high-quailty licorice.In this paper,HPLC was used to assay the contents of four flavonoids,liquiritin,isoliquiritin,liquiritigenin,and isoliquiritigenin in 60 licorice samples.CHI cDNA was amplified from 6 licorice samples as the group with high flavonoids contents and 5 licorice samples as the group with low flavonoids contents by RT-PCR,and the major haplotypes of the groups with high/low flavonoids contents were determined.The bioinformations of the major haplotypes were analyzed by bioinformatics tools.The recombinant expression vectors,pPIC9K-CHI,were constructed and transformed into disarmed Pichia pastoris GS115 by electroporation.The expression of CHI was induced by methanol and detected by SDS-PAGE.The CHI protein was used for enzymatic reactions and the contents of products were assayed by HPLC.The results of this paper are as follows:(1)CHI cDNA cloning and polymorphism results:113 CHI cDNA sequences with a full length of 690 bp were obtained encoding 229 amino acid residues.97 variable sites were found in the 113 CHI cDNA sequences,53 haplotypes(haplotype 1?53)were determined,and the similarity of which was 99.64%.63 variable sites were found in the 113 CHI sequences,44 amino acid sequence types(AA-1?AA-44)were determined,and the similarity of which was 99.10%.AA-1 was encoded by haplotype I?7 and AA-30 was encoded by haploytype 36 ? 39.Haplotype 1 is the major haplotype in the group with high flavonoids contents(30 sequences,45.45%),while haplotype 36 is the major haplotype in the group with low flavonoids contents(25 sequences,53.19%).The mutation site of haplotype 1 and haplotype 36 is C/A at 246 bp.(2)Bioinformatics analysis results:The physicochemical property results of AA-1 are similar to that of AA-30.The secondary structure analysis result of AA-1 was same as AA-30 has.The two CHI amino acid sequences have the same tertiary structure analysis results,and the mutation site(246 bp)was not consisted in the binding area with substrates.The CHI protein contains no signal peptide and belongs to a chalcone 3 superfamily domain.And the CHI protein is entirely outside the cell membrane indicating that there is no transmembrane domain in the CHI protein.Homology analysis indicated that CHI had a clear discrimination between different species.(3)The results of heterologous expression in P.pastoris GS115:Two recombinant P.pastoris GS115 strains,G-P-CHI-1 and G-P-CHI-36,which contained the major haplotype in the group with high/low flavonoids contents were constructed,respectively.After a series of selections including His+ transformant selection,G418 selection,Mut+ transformant selection,PCR and sequencing verification,the above two recombinant P.pastoris GS115 strains were successfully constructed.Methanol was used as inducer in the expression of the two recombinant strains.The SDS-PAGE result demonstrated the right expression of CHI,which obtained a 24 kDa protein same as the size of CHI.(4)The results of vitro enzymatic reaction:Isoliquiritigenin and naringenin chalcone were used as the substrate of enzymatic reactions,respectively.And the expressed CHI proteins were used as enzyme.HPLC results indicated that the catalytical activitiy of CHI-1 was higher than that of CHI-36.It demonstrated that the cataytical activity of the major haplotype in the group of high flavonoids contents was higher than the cataytical activity of the major haplotype in the group of low flavonoids contents.