| Steroidal saponins,the main active compounds in Dioscorea plants,usually show wide range of pharmacological effects,and their biological activity is closely related to the chemical structure.Different glycosyl side chains have a great influence on their activity.In Dioscorea zingiberensis,the diosgenin 3-O-glycosyltransferase(DzS3GT)catalyzes the glycosylation of diosgenin to trillin;furostanol glycoside 26-O-β-glucosidase(DzF26G)catalyzes the conversion of furostanol saponins to spirostanol saponins,they two are the key enzymes involved in the biosynthesis and conversion of steroidal saponins.The study about the key enzymes/genes and metabolic regulation mechanism of steroidal saponins may be helpful to indicate how to regulate the synthesis of steroidal saponins and provide a theoretical basis for steroidal saponin biosynthesis in Dioscorea plants for crop improvement via genetic engineering.By RT-PCR and RACE method,the full-length cDNA of DzS3GT was cloned.The ORF of DzS3GT gene is 1731 bp,encoding for 577 amino acids.The phylogenetic tree analysis showed that DzS3GT had higher affinity with AsSGT and WsSGTL1,which belonged to the enzymes that mainly transfer the glucosyl other than those can transfer different kinds of sugar donor.The substrate docking model using DzS3GT and diosgenin predicted that the amino acid Asp-144 forms hydrogen bonds with the 3-OH position of diosgenin.The DzS3GT gene was prokaryotic expressed by E.coli BL21(DE3).The pEASY-DzS3GT fusion protein was about 70kDa,and can transfer a glycosyl from UDP-glucose to the 3-OH position of diosgenin.The optimal glycosyltransferase activity of rDzS3GT was obtained at 50℃ and at pH 6.5.The enzyme was strongly inhibited by Mg2+,Fe3+ and Cu2+ ions.The C-terminal and N-terminal RFP fusion expression vectors of DzS3GT were constructed.The subcellular localization result showed that DzS3GT was mainly located in the cytoplasm.The relationship between DzS3GT gene expression,DzS3GT activity and accumulation of saponin in leaves,stems and rhizomes of Dioscorea zingiberensis was studied.The results showed that DzS3GT mainy active in leaves.The trillin content was much higher in the leaves than in other organs.The gene expression and saponins accumulation suggest that the biosynthesis of trillin mainly in the leaves of D.zingiberensis.This study provides a potential relevance to the biosynthesis and transport mechanism of steroidal saponins in Dioscorea plant.We speculate that diosgenin is converted to trillin in the leaves and subsequently be glycosylation to other saponins for easier transportation or storage.A rapid and simple HPLC-MS method for simultaneous determination of eight steroidal saponins in different processed D.zingiberensis rhizomes and several other medicinal plants was developed.Hot-air drying technology at 60℃ could be used to gain more spirostanol saponins.Wine-processing technology could be used to kill the enzymes in D.zingiberensis for gaining more furostanol saponins with better solubility in water.These results might be helpful for further studies on the steroidal saponins conversion mechanism.By RT-PCR and RACE method,the full-length cDNA of DzF26G was cloned.The ORF of DzF26G gene is 1641 bp,encoding for 547 amino acids.The DzF26G gene was prokaryotic expressed by E.coli BL21(DE3).The pEASY-DzF26G fusion protein was about 60kDa,and can catalyze the hydrolysis of furostanol saponins into spirostanol saponins.We have studied the relationship between DzF26G expression and the accumulation of saponins in different organs of D.zingiberensis during different developmental stages.The result showed that the change trend of DzF26G gene expression was consistent with that of saponins content and DzF26G gene expressed mainly in the leaves.However,the substrate of the DzF26G enzyme is accumulated mainly in the rhizomes,indicating that the expression of DzF26G may be regulated at transcription and translation level.The DzF26G enzyme was isolated and purified from the rhizome of D.zingiberensis with 73.23-fold purification and a specific activity of 260.74U/mg.The molecular weight of DzF26G was about 60kDa.The optimal pH and temperature of DzF26G withρNP-β-D-Glc as substrate were 6.0 and 50℃.It was strongly inhibit by Fe2+,Fe3+ Cu2+,Hg2+ metal ions.The chemical modification agent NBS and TNBS significantly inhibited the activity of DzF26G,indicating that tryptophan and lysine may be the necessary groups for the DzF26G catalytic activity center;DEPC and DIC have obvious activation effect on DzF26G,indicating that histidine and arginine residues may be involved in the catalytic reaction of DzF26G;DTT has little effect on the activity of DzF26G,indicating that the sulfur group may not be a necessary group of DzF26G activity.DzF26G can hydrolyzeρNP-β-D-Glc as well as ρNP-β-D-Gal and ρNP-β-D-Rha.The catalytic capacity of DzF26G for hydrolyzing furostanol saponins from strong to weak were:protodioscin,parvifloside,protodeltonin,protobioside.Finally,the mechanism of the transformation of steroidal saponins in the drying process was studied.The relationship between DzF26G activity,saponins content and DzF26G expression was studied during the drying process of D.zingiberensis rhizomes.It was further demonstrated that DzF26G enzyme was involved in the in vivo conversion of steroidal saponins.In addition,we prepared the polyclonal antibody of DzF26G protein and the monoclonal antibody of parvifloside.The preliminary immunohistochemistry results showed that the enzyme and substrate were located in vascular bundles and parenchyma cells at the same time.DzF26G subcellular localization studies have shown that DzF26G is mainly located in the cytoplasm.We speculate that the steroidal saponins of D.zingiberensis accumulate in the vacuoles of the cells,and DzF26G is distributed in the cytoplasm.When the cell environment changes(such as drying),the cell compartment may be destroyed,then the enzyme and the substrate are in contact.Thus,the catalytic reaction takes place. |
