A New Molecular Mechanism Regulating The Biosynthesis Of Glycyrrhizic Acid-functional Gene CNVs

Glycyrrhiza uralensis Fisch.is one of the most-frequently-used Chinese herb for its effects of nourishing qi,alleviating pain,tonifying spleen and stomach,eliminating phlegm and relieving coughing.Due to recent years’ excessive harvest,wild resources of G.uralensis are now rare and turning endangered.In 2000,the Chinese government imposed restrictions on the collection of wild G.uralensis plants.As a result,cultivation has become the main source of G.uralensis supply.However,the content level of glycyrrhizic acid in most of the G.uralensiscultivars fails to meet the requirement of Chinese pharmacopoeia.Therefore,improving the quality of G.uralensiscultivars has become a key issue to restrict the sustainable development of G.uralensis resources.Among the components in G.uralensis,glycyrrhizic acid is believed to be the marker active compound.Previous studies have shown that glycyrrhizic acid possesses various biological activities,such as anti-inflammatory,antitumor and immune-stimulating activities.Up to date the biosynthesis of glycyrrhizic acid through mevalonic acid(MVA)pathway has been clarified.Three key enzymes in MVA pathway are 3-hydroxy-3-methylglutary Co A reductase(HMGR),squalene synthase(SQS)and beta-amyrin synthase(β-AS).HMGR is the first rate-limiting enzyme in this pathway,which catalyzes irreversible reaction of HMG-CoA and NADPH into MVA.SQS is situated in a branch point and may be an up-regulator for production of triterpenoids.Beta-AS catalyzes 2,3-oxidosqualene into beta-amyrin,an important precursor of glycyrrhizic acid.Copy number variations(CNVs),a new type of genomic variation that has recently received considerable attention,are deletions,insertions,duplications,and more complex variations ranging from 1 kb to submicroscopic sizes.Reports have linked human CNVs to diseases.We have found previously that the functional genes have copy number variations in G.uralensis.Based on central dogma of molecular biology,it is well known that the difference in copy number of gene can lead to content variations of the relevant enzyme,and finally influence the accumulation of products.Therefore,CNVs of the three functional genes can lead to content variations of the relevant enzyme,and finally influence the accumulation of glycyrrhizic acid in G.uralensis.In this paper,the relationship between functional gene CNVs and the accumulation of glycyrrhizic acid was analyzed at three different levels,natural G.uralensis plants,yeast expression system and transgenic G.uralensis regeneration plants,based on the three functional genes,HMGR,SQS1 and β-AS involved in biosynthesis pathway of glycyrrhizic acid.We tried to reveal how the functional genes control and regulate the secondary metabolic pathway in G.uralensis and develope genetically modified G.uralensiscultivars with enhanced glycyrrhizic acid accumulation.And we believe that it will be helpful for producing glycyrrhizic acid by biotechnology and relieving the deficit of glycyrrhizic acid resource.The following conclusions were drawn in this paper:(1)At the whole plant level,it was confirmed that functional gene CNVs influence the accumulation of glycyrrhizic acid.Among six CNVs groups,group B with two copies of HMGR,one copy of SQS1 and β-AS contained relatively higher contents of glycyrrhizic acid.The accumulation of glycyrrhizic acid was lower in the group C with two copies ofβ-AS,one copy of SQSl and HMGR.All of the samples in group A and B were collected from genuine producing areas of G.uralensis,and 2/3 smples in group C were collected from other areas.But functional gene CNVs can not influence the external morphology and internal structure significantly.(2)The relationship analysis between functional gene CNVs and external morphology and microscopic structure showed that functional gene CNVs did not obviously influence the shape indexes,such as plant height,diameter,bark color,and the microscopic structure indexes,such as the thickness of palisade tissue and spongy tissue.However,as far as the number of gland place on leaves was concerned,group B had the most.Because group B also contained relatively higher contents of glycyrrhizic acid,it was speculated that there was a certain correlation between the number of gland place on leaves and the content of glycyrrhizic acid.(3)At the recombinant yeast level:It was found that the contents of ergosterol in all recombinant Pichia.pastoris containing SQS1 and HMGRwere higher than that in negative control.And with the increase of copy number of functional genes,the contents of ergosterol showed an increasing-decreasing-increasing pattern.(4)A new technology using functional gene CNVs for analyzing the varying pattern of active constituent in medicinal plant was created.First,the copy number of functional genes in metabolic pathway in this medicinal plant was detected.Second,the content of metabolites was assayed.Finally,the relationship between them was analyzed.(5)The root specific expression vectors containing three functional genes of G.uralensis,respectively,were constructed and transferred into G.uralensis cells successfully.Genetically modified G.uralensisplants with over-expressed HMGR,SQS1 and β-AS genes were obtained.The results of this work are significant for further studies to reveal the molecular mechanism of glycyrrhizic acid biosynthesis and enhance glycyrrhizic acid accumulation in G.uralensis.