Research On The Function Of Licorice CHS And CHI Genes Based On Hairy Root Culture System

Licorice is one of the most frequently used Chinese herbs and known as the“Shi Fang Jiu Cao".In recent years,the commonly present quality degradation and low content of active ingredients in cultivated licorice has become a key issue restricting the sustainable development of licorice resources.A large number of reports have shown that hairy roots can stably and efficiently produce secondary metabolites of medicinal plants.Therefore,it is of great value to investigate the secondary metabolic pathways in licorice by using hairy root culture systems.Liquiritin is one of the most important active compounds in licorice.Chalcone synthase(CHS)and chalcone isomerase(CHI)are two key rate-limiting enzymes involved in the liquiritin biosynthetic pathway.Therefore,based on the previously cloning of CHS and CHI genes in G.uralensis,this study aims to investigate the function of CHS and CHI genes by hairy root culture systems,and analyze their influence on the biosynthesis of flavonoids.The results of this paper will also contribute to screening the hairy root system with high level accumulation of flavonoids,and lay a theoretical foundation and provide technical support for the production of licorice flavonoids in vitro.Specific CHS and CHI haplotypes were selected according to the analysis of the relationship between the contents of flavonoids and the gene polymorphisms of CHS and CHI.Plant binary expression vectors containing specific CHS and CHI genes were constructed by gene fusion,and then transformed into Agrobacterium rhizogenes.The explants of G.uralensis were infected by the above recombinanted A.rhizogenes to induce hairy roots,and which were verified by PCR and sequencing,UPLC method was established to simultaneously assay the contents of the four main flavonoids in transgenic hairy root samples.The copy numbers of target genes in transgenic hairy root samples were determined by qRT-PCR.And the relationships between the copy numbers of the target gene and the content of flavonoids in the hairy roots were analyzed.The results of this paper are as follows:(1)According to the analysis of the relationship between the contents of flavonoids and the gene polymorphisms of CHS and CHI,the specific CHI-1(GenBank Accession number:KY115232)and CHS-60(GenBank Accession number:KY810356)corresponding to the high content of flavonoids were selected.(2)CHI-1 and CHS-60 were successfully inserted into pCAMBIA1305.1 at sites Bgl ? and Spe I by gene fusion method,and the plant binary expression vectors,pCA-CHI and pCA-CHS,were constructed.(3)pCA-CHI and pCA-CHS were transformed into A.rhizogenes by electroporation,and the recombinanted A.rhizogenes were obtained.(4)Explants of G.uralensis were infected by the above recombinanted A.rhizogenes to induce hairy roots,which were verified by PCR and sequencing.(5)An UPLC method to assay the four main flavonoids in licorice hairy roots simultaneously was established and used to analyze the contents of four flavonoids in 54 transgenic G.uralensis hairy root samples.The statistical analysis showed that the difference in the content of flavonoids between the transgenic hairy roots overexpressing CHS gene and wild hairy roots,and the transgenic hairy roots overexpressing CHI gene and wild hairy roots is significant.By comparing the rank mean,the content of flavonoids in the transgenic hairy roots overexpressing CHI gene was the highest,and in the wild hairy roots was the lowest,which confirmed that overexpression of CHS and CHI genes enhanced the content of flavonoids in hairy roots.(6)qRT-PCR was used to analyze the copy number of target genes in 15 transgenic G.uralensis hairy roots.The copy number of CHI gene in 8 transgenic G.uralensis hairy roots was 1,2,or 5,respectively.And the copy number of CHS gene in 7 transgenic G.uralensis hairy roots was 7,9,10,11,13 or 18,respectively.(7)According to the relationship analysis between gene copy number and the content of flavonoids,the transgenic hairy root lines with 9 copies of CHS gene and 5 copies of CHI gene were finally selected as the hairy root materials for further large-scale culturing and flavonoids accumulation in vitro.