| Licorice is one of the most-frequently-used Chinese herbs with the effects of nourishing qi,alleviating pain,tonifying spleen and stomach,eliminating phlegm and relieving coughing.Because the exploitation of wild licorice was prohibited by Chinese government,licorice cultivars have gradually become the mainstream commodity in Chinese herbal medicine market.However,our previous investigation showed that the content of glycyrrhizic acid(GA)in cultivars was generally low and difficult to meet the minimum requirment(2.0%)stipulated in the Chinese Pharmacopoeia,which severely influenced the quality of cultivated licorice and the efficacy of Chinese patent medicine containing licorice.GA is the most important active compound in licorice and one of the marker components stipulated in the Chinese Pharmacopoeia.It is a pentacyclic triterpenoid compound synthesized via the mevalonic acid(MVA)pathway,which is regulated by many key enzymes.Among them,?-amyrin synthase(?-AS)catalyzes the 2,3-oxidosqualene to form ?-amyrin.It plays an important role to biosynthesize the pentacyclic triterpene skeleton and GA finally.Therefore,the study of the gene polymorphism of ?-AS gene will be helpful to clarify the molecular mechanism of GA biosynthesis and provide a theoretical basis for improving the quality of cultivated licorice.The reason why functional gene polymorphism causes the difference in secondary metabolite content mainly exists in two aspects.First,gene polymorphism leads to the difference in gene expression level.On the other hand,gene polymorphism leads to the difference in enzyme structure and function.Therefore,this paper proposed the hypothesis that "functional gene-enzyme mutation is the molecular mechanism in GA biosynthesis",and performed progressive analysis from two aspects,functional genes and enzymes.First,the polymorphism of ?-AS gene was analyzed and the specific ?-AS genotypes corresponding to the low/high content of GA were screened.Then,further analysis of gene and enzyme expression was performed.This paper is important for the molecular breeding of high quality licorice and the guidance of similar studies in other medicinal plants.The following methods were used in this paper:the content of GA in different licorice samples from three origins and five different habitats was assayed by HPLC.Using reverse transcription PCR,?-AS gene was cloned from the root of licorice and the gene polymorphism was analyzed.The specific ?-AS genotypes corresponding to the high/low content of GA were screened,respectively.Using qRT-PCR,the relative expression of different ?-AS genes was analyzed.The recombinant Pichia pastoris GS115 containing the specific ?-AS genotypes were constructed respectively,and induced by methanol.The following results were got in this paper:(1)The content of GA in 60 licorice samples from three different origins and five different habitats was assayed by HPLC.Overall,there was a significant difference in the content of 18?-GA among licorice samples from three different origins.The content of 18?-GA in Glycyrrhiza uralensis samples was the highest,in Glycyrrhiza inflate samples was the lowest,and in Glycyrrhiza glabra samples was between the above two.There was no significant difference in the content of 18a-GA between samples of G.uralensis and G.glabra,but there was a significant difference in the content of 18?-GA between samples of G.uralensis and G.inflata,and G.inflata and G.glabra.The content of 18a-GA in G.uralensis samples was the highest,in G.inflata samples was the lowest,and in G.glabra samples was between the above two.In addition,the correlation analysis showed that there was a positive correlation between the content of 18a-GA and 18?-GA in all samples.According to the above results,four groups,Group 1(18a-GA was not detected),Group 2(with a high content of 18a-GA),Group 3(with a low content of 18?-GA),and Group 4(with a high content of 18?-GA)were determined for further analysis of the ?-AS gene polymorphism.(2)Thirteen ?-AS cDNA sequences with a full length of 2289 bp were obtained,and eleven haplotypes were determined,which encoded five amino acid sequences with a full length of 762 amino acids.The results of gene polymorphism analysis showed that specific variation sites at 104 bp(G-C)and 106 bp(G-C)were present in?-AS cDNA sequences of Group 1 and 3,which led to the variation of serine-threonine(S-T)transversion and alanine-proline(A-P)transversion at 35 and 36 amino acid residues,respectively.35-T and 36-P were specific in Group 1 and 3,while 35-S and 36-A were specific in Group 2 and 4.(3)The relative expression of different ?-AS gene in four groups was analyzed.There was an obvious difference in the expression of ?-AS gene among different samples.The relative expression of ?-AS gene in Group 3 was higher than that in Group 1,and in Group 4 is higher than that in Group 2,which indicated that there was a positive correlation between the high level of GA and the high level of ?-AS gene expression.(4)Recombinant P.pastoris GS115,GS115-pPIC9K-BAS-3 and GS115-pPIC9K-BAS-8,containing specific ?-AS genotypes were successfullyconstructed.His+transformant selection,geneticin G418 selection,Mut+recombinants selection,PCR validation and sequencing validation were performed with GS115-pPIC9K-BAS-3 and GS 115-pPIC9K-BAS-8.Methanol was used to induce the expression of GS 115-pPIC9K-BAS-3 and GS 115-pPIC9K-BAS-8,which was detected by SDS-PAGE.Results showed that a 87 kDa protein was obtained,which was consistant with the size of ?-AS.This paper will lay a foundation for analyzing the molecular mechanism in GA biosynthesis and improving the quality of cultivated licorice,and also provide a theoretical basis for superior genotypes screening and molecular breeding of licorice. |
PDF Full Text Request