Functional Study Of Panax Notoginseng Glycosyltransferase Genes PnUGT6350，PnUGT3498 And PnUGT4291

Panax notoginseng is a precious traditional medicinal herb belonging to the Araliaceae family.It contains mainly contains triterpene saponins as its medicinal components.And these substances are synthesized through a series of key enzyme genes through two metabolic pathways: the mevalonate pathway(MVA)and the methylerythritol phosphate pathway(MEP).In this study,based on the transcriptome analysis of P.notoginseng adventitious roots that treated by methyl jasmonic acid,we screened for potential glycosyltransferase genes that are associated with triterpene saponin synthesis: PnUGT6350,PnUGT3498 and PnUGT4291.And the function of these genes in triterpene biosynthesis were investigated by bioinformatic analysis and transgenic approaches.The specific research results are as follows:1.Three glycosyltransferase genes related to triterpenoid biosynthesis were screened from the transcriptome data of adventitious root of P.notoginseng treated with methyl jasmonic acid,respectively PnUGT6350,PnUGT3498 and PnUGT4291.And these genes belonged to the UGT73 and UGT74 families,and the predicted modification site was C3.The PnUGT6350,PnUGT3498 and PnUGT4291 genes were cloned from the c DNA of P.notoginsengadventitious roots.And the lengths of three genes were 1482 bp,1431 bp and1368 bp,respectively,and encoding proteins of 493,476 and 455 amino acids,respectively.Bioinformatic analysis revealed that all three glycosyltransferase proteins belong to hydrophilic proteins,without transmembrane domains and signal peptides,and contain conserved domains of glycosyltransferase,belonging to the glycosyltransferase family.The specific expression of PnUGT6350,PnUGT3498 and PnUGT4291 genes in different tissues,including adventitious root main root,adventitious root lateral root,tissue culture seedling root,petiole and leaf was analyzed.The results showed that all three genes were expression in the five tissues.Among them,the expression level of PnUGT6350 and PnUGT3498 genes in adventitious root main root were significantly higher than in other tissue,while the expression level of PnUGT4291 gene as the highest in the leaf.2.The pROKⅡ-PnUGT6350,pROKⅡ-PnUGT3498 and pROKⅡ-PnUGT4291 plant expression vectors were successfully constructed.The pROKⅡ-PnUGT6350 and pROKⅡPnUGT3498 genes were transferred into Nicotiana tabacum by leaf disc transformation method,and 8 strains of transgenic Nicotiana tabacum with PnUGT6350 gene were obtained,of which the higher expression was No.10 and No.14 strains;ten PnUGT3498 transgenic Nicotiana tabacum lines were obtained,among which No.4 and No.5 had higher expression level.The four transgenic Nicotiana tabacum lines with high expression levels were subjected to feeding with the precursor of oleanolic acid and provided substrates for glycosyltransferase,and the calendula glycoside E was detected as target product.The results showed that PnUGT6350 transgenic Nicotiana tabacum No.10 and No.14 produced a high content of calendulaside E,while the PnUGT3498 transgenic Nicotiana tabacum No.4 and No.5 did not detect calendulaside E.3.The transient transformation system of P.notoginseng adventitious roots was established,and PnUGT6350,PnUGT3498 and PnUGT4291 genes were transiently transferred into adventitious roots.The contents of five saponins(calendulaside E,R1,Rg1,Re and Rb1)in the adventitious roots of transgenic PnUGT6350 were determined,which were 1.38 times,1.75 times,2.42 times,1.72 times and 2.29 times of the control,respectively.The contents of calendulaside E,R1,Rg1,Re,and Rb1 in the adventitious roots of transgenic PnUGT4291 gene were determined.The contents of R1,Rg1 and Rb1 were 1.56 times,2.04 times and 1.83 times that of the control,respectively,while the contents of calendulaside E and Re did not change significantly.And the contents of calendulaside E,R1,Rg1,Re and Rb1 in the adventitious roots of PnUGT3498 gene were 1.27 times,1.23 times,1.64 times,1.30 times and1.79 times of the control,respectively.The stable genetic transformation system of P.notoginseng adventitious roots was established.And the PnUGT6350 and PnUGT3498 genes were successfully transferred into the adventitious roots,and 20 transgenic adventitious root lines with PnUGT6350 gene and 8 transgenic adventitious root lines with PnUGT3498 gene were obtained,respectively.