| Panax japonicus C.A.Mey.var.major(Burk.)is a species of the Panax genus in the Araliaceae family,with a long history of medicinal use in China.It is believed to have effects such as nourishing the lungs and kidneys,promoting blood circulation,relieving pain,and stopping bleeding.The main active components of P.japonicus are triterpenoid saponins,including dammarane-type and oleanane-type saponins.P.japonicus is a perennial herb that requires 3-6 years to grow before it can be used for medicine,and its cultivation process requires crop rotation,resulting in low land utilization and a long-standing supply-demand imbalance.To address the shortage of medicinal resources caused by this high-value,low-yield medicinal plant,establishing a homologous or heterologous high-efficiency expression system(synthetic biology)using modern biotechnology to directly produce medicinal active ingredients is an important potential technological approach.Although the use of synthetic biology technology to produce P.japonicus saponins has promising applications,the premise for conducting such research is to have a detailed understanding of the saponin biosynthesis pathway in order to flexibly use relevant gene components and construct high-efficiency expression systems for saponins.Currently,the exploration and functional research of genes related to P.japonicus saponin biosynthesis are not comprehensive enough,especially the biogenesis pathway of ginsenoside Rb2,a dammarane-type saponin found in P.japonicus,is not well understood and requires further investigation.In this study,RNAi technology was used to downregulate the expression of key enzyme genes Pn UGT3 and Pn3-32-i5 that control the synthesis pathway of ginsenoside Rg3 in P.japonicus cells,thereby altering the saponin biosynthesis metabolic flux in P.japonicus cells and affecting the synthesis of ginsenoside Rb2.Through transcriptome sequencing,differentially expressed genes were identified and combined with literature reports and functional gene research results to explore the glycosyltransferase genes related to the synthesis of ginsenoside Rb2 in P.japonicus,deepening the understanding of the biosynthesis pathway of P.japonicus saponins.The specific research results are as follows:This study used RNAi technology to reduce the expression of key enzyme genes Pn UGT3 and Pn3-32-i5,which control the synthesis pathway of ginsenoside in P.notoginseng cells,thereby changing the metabolic flow of ginsenoside synthesis in the cells and affecting the synthesis of ginsenoside Rb2.Through transcriptome sequencing,the study identified sugar transferase genes related to the biosynthesis of ginsenoside Rb2 in P.notoginseng by mining gene expression differences,combined with literature reports and functional studies of genes.Specifically,RNAi fragments of Pn UGT3 and Pn3-32-i5 genes were amplified from P.notoginseng c DNA and integrated into the genome DNA of P.notoginseng cells by Agrobacterium-mediated transformation using the constructed RNAi expression vectors p Hellsgate-Pn UGT3-i and p Hellsgate-Pn3-32-i5-i.After resistance gene screening and molecular identification,three positive P.notoginseng cell lines with decreased expression of Pn UGT3 and Pn3-32-i5 genes were obtained,respectively.Transcriptome sequencing was performed on these positive cell lines,and the sequencing data were analyzed for functional annotation,gene expression level analysis,differential expression analysis,gene set analysis,and gene structure analysis.Two candidate sugar transferase genes possibly involved in the biosynthesis of ginsenoside Rb2 in P.notoginseng,Pj UGT1 and Pj UGT2,were preliminarily screened.Furthermore,the functional analysis of the candidate genes was conducted using the prokaryotic expression system.Two prokaryotic expression vectors,p ET32 aPj UGT1 and p ET32a-Pj UGT2,were constructed and transformed into Escherichia coli DE3.The catalytic function of the target enzyme protein was explored by constructing an enzyme reaction system.The results showed that Pj UGT1 had the function of catalyzing the first glucose glycosylation at position C-3 of ginsenoside Rb2,converting protopanaxadiol(PPD)to ginsenoside Rh2 and ginsenoside CK to ginsenoside F2.In addition,Pj UGT2 had the function of catalyzing the second glucose glycosylation at position C-3 of ginsenoside Rh2,further converting ginsenoside Rh2 to ginsenoside Rg3.The above research results have improved some steps in the biosynthesis pathway of ginsenoside in P.japonicus,providing a basis and reference for the production of related important monomeric saponins using synthetic biology techniques. |
