Exploration And Functional Study Of UDP-Rhamnose Synthase Gene From Caragana Korshinskii

L-Rhamanose is also known as 6-Deoxy-L(+)-mannose,Widely present in plant polysaccharides,glycosides,plant pectins and bacterial polysaccharides,It is also an important component of many bacterial,plant,and fungal cell walls.With the continuous elu Cidation of the biomedical activities and pharmacological mechanisms of rhamnoside compounds,Rhamnoside compounds have attracted more and more attention.UDP-rhamnose,as a direct glycosyl donor for the synthesis of rhamnoside,parti Cipates in the formation of various rhamnosides,and is very important in the synthesis of rhamnoside.UDP-rhamnose is Conjugation with secondary metabolites such as flavonoids and terpenoids under the catalysis of glycosyltransferase,produces soluble rhamnoside and exerts its spe Cial biological activity and pharmacological properties.However,commer Cial UDP-rhamnose is difficult to obtain,chemical synthesis is very cumbersome and there are too many by-products,which greatly limits the biological research related to plant rhamnoside.Therefore,the biosynthetic pathway of UDP-rhamnose has attracted much attention and is a hot spot in the field of flavonoids in recent years.Caragana chinensis,a characteristic medi Cinal plant of Inner Mongolia,was used as material in this study.From its genomic(unpublished)data,4 UDP-rhamnose synthase genes were mined by Blast analysis and alignment,they were named as Ci RHM-1,Ci RHM-2,Ci RHM-3 and Ci NES/ER.Bioinformatics analysis of Ci RHM as a trifunctional enzyme with oxidative,isomerized and reduced functions,UDP-rhamnose could synthesized directly from UDP-glucose in one step.While Ci NRS/ER is a bifunctional enzyme,it can only have the functions of isomerization and reduction.After that,the four genes were amplified by PCR to construct a prokaryotic expression vector,and expressed in E.coli expression system.The results showed that the prokaryotic expression of Ci RHM-1 as inclusion bodies,Water-soluble proteins that can be expressed and obtained by Ci RHM-2 and Ci NRS/ER,SDS-PAGE electrophoresis showed that the purity was above 95%.However,Ci RHM-3 could not be expressed in prokaryotic expression system whether using Mbp tag or His tag.In order to further study,the catalytic mechanism of rhamnose synthase,we purified Ci RHM-2 and Ci NRS/ER enzyme proteins using nickel columns,ion columns and molecular sieves,Finally,the protein crystals were screened with eight crystallization kits from Hamptons.