| Lignans constitute one of themajor chemical classes with antiviral activities in Isatis indigotica Fort.Currently,lignan compounds identified from I.indigotica include the following four classes: ditetrahydrofurans(pinoresinol,pin),tetrahydrofurans(lariciresinol,lar),diaryl butanes(secoisolariciresinol,sec),diaryl butyllactones(matairesinol,mat)and their glycoside derivatives.Modern pharmacological studies have shown that glycosidic group is important structure of the compounds lignans antiviral activity,and different forms of glycosidation also lead to differences in antiviral activity,and the glycosidation of lignans in plants is catalyzed by glycosyltransferases(uridine diphosphate-dependent glycosyltransferases,UGT),but the glycosyltransferases that catalyzes lignans glycosidation in I.indigotica has not been reported.First,Genome wide gene annotation was carried out for the prediction of UGT gene family in I.indigotica.An omologous phylogenetic tree based on putative UGTs proteins was constructed for the further functional prediction.Two UGT71B5 s were clustered with AtUGT72B1,which catalyzes glycosylation of monolignol,and were supposed to catalyze lignans.Subsequently,the function of UGT was verified by in vitro enzyme activity experiment.The results showed that UGT71B5-1 could catalyze the monoglycosidation of pinoresinol and lariciresinol,as well as the production of pinoresinol diglycosides.UGT71B5-2 only catalyzed the monoglycosidation of pinoresinol with low efficiency.Secondly,LC-MS was used to characterize the spatial distribution of lignans in different organs(root,leaf)and root tissues(epidermis and cortex,phloem,xylem and cambium)of I.indigotica.It was found that I.indigotica contained at least 4 lignans and 4 lignan glycosides,and most lignans existed as glycosides.Pinoresinol,pinoresinol 4-O-glucoside and matairesinol monoglucoside were mainly distributed in roots,and the content was the highest in epidermis and cortex.The content of lariciresinol,secoisolariciresinol and secoisolariciresinol monoglucoside in leaves was much higher than that in roots.Then,through the study on the physicochemical properties of UGT71B5-1,it was found that UGT71B5-1 had the highest activity at pH 8.0 and temperature of 30?,and it was a non-metal cationic dependent protease,which optimized the conditions for the subsequent enzymatic reaction.Next,the catalytic characteristics of UGT71B5-1 were further investigated,and promiscuity of UGT71B5-1 towards a wide range of lignan substrates and sugar donor was found.Meanwhile,ditetrahydrofuran lignans(such as pinoresinol)represented as the optimal substrate for UGT71B5-1.Finally,transcriptional traits of two UGTs were studied,the result is expressed as that UGT71B5-1 was both expressed in roots and leaves,and the expression level in the leaves was much higher than that in roots.To further exam its tissues transcriptional traits in root,the expression of epidermis and cortex was the highest,and the difference between xylem and cambium and phloem with low expression was not significant.At the same time,the results of subcellular localization using fusion fluorescent protein showed that UGT71B5-1 is expressed in the cytoplasm,which is consistent with most reported UGT.In addition,the catalytic characteristics and expression characteristics of UGT71B5-2 are similar to that of UGT71B5-1,but the catalytic activity is far lower than that of UGT71B5-1 and the relative expression of the gene is also lower.In summary,this study investigated two UGTs with glycosidation activity of pinoresinol,and analyzed their enzyme catalytic properties and gene expression characteristics,which provided references for the subsequent discovery of UGTs and catalytic elements for the metabolic engineering of lignan glycosides. |
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