Studies On Adventitious Roots Culture Of Panax Notoginseng And Ginsenosides Biosynthesis & Regulation

Panax ginseng and Panax notoginseng are famous bulk medicinal Panax plants.Ginsenosides and notoginsenosides are the main pharmacologically active ingredients.They are widely used in medicine,but also in health care products,daily chemical products,etc.with huge market demand.Field cultivation is their main source,which has serious problems of long growth cycle,requiring a lot of manpower and material resources,continuous cropping obstacles,pesticide residues and heavy metal pollution.The development of plant biotechnology production provides a new supply mode for the production of active ingredients in P.ginseng and P.notoginseng.Plant biotechnology production has advantages of short growth cycle,artificially controlled cultivation conditions and could get rid of natural geographical environment and seasonal restrictions.The research group has been engaged in studying the biotechnology production of medicinal plants for a long time and successfully obtained the tissue culture system of P.ginseng,P.quinquefolius,Glycyrrhiza uralensis,Astragalus membranaceus,etc.The adventitious roots（ARs）culture system of P.ginseng has been industrialized in Dalian Practical Biotechnology CO.,LTD.and won the second prize of National Technological Progress Award.Based on the problems in cultivation and advantages of plant biotechnology production in the research group,this thesis carried out research on the induction and screening of ARs of P.notoginseng and bioreactor optimization,in-depth study on mining glycosyltransferases involved in ginsenoside biosynthesis from P.ginseng and P.notoginseng,the regulation mechanism of ginsenoside biosynthesis and the construction of a high-yield system of ginsenoside Rg₃ in plant chassis.First,the adventitious roots of P.notoginseng were induced and optimized.The ARs were successfully induced from callus.The subculture medium for ARs were systematicallyoptimized as 3/4 SH+3%sucrose+1 mg/L naphthylacetic acid+3mg/L indole-3-butytric acid+0.1 mg/L kinetin,p H=5.6.The cultivation of P.notoginseng ARs in balloon-type bubble bioreactor was also performed from inoculum density,cultivation cycle and adding medium time,respectively.The optimal inoculum density（2%）,cultivation cycle（40 days）and adding medium time（25th day）were more beneficial to biomass accumulation.The adventitious rootsof P.notoginseng were screened.During the cultivation process,the ARsofP.notoginsenggenerated three different morphologies,multi-branched（MB）root,thin-long（TL）root and thin-short（TS）root.By microscopic comparsion,MB root had similar structure to that offield cultivated roots,except for the no branch fibre.The structure morphology in three adventitious roots hasno difference.MB root also showed close gene expression levels and metabolite profiles,which were similar to those offieldcultivated roots.MB root had a high yield of total saponins with 17.92 mg/g,which has the potential in lagre scale cultivation.The elucidation of the biosynthetic pathways of active ingredients is the basis of synthetic biology.At present,the biosynthetic pathways of notoginsenosides and ginsenosides are not very clear and the key enzyme activities are not high.Therefore,comparative transcriptomic profiling ofP.ginseng and P.notoginsengwas performed toscreen the keygenes directly involved inginsenosidesbiosynthesis.Combination the screening of conservativePlant Secondary Product Glycosyltransferasemotif and phylogenetic analysis,13 of potential UGT genes involved in ginsenosides biosynthesis were discovered.The correlation between these UGT genes and ginsenosidescontent discovered that Cluster-26044.20938,Cluster-26044.15116,Cluster-26044.5920 could be related to the accumulation of total saponins,PPD,and PPT.Cluster-32220.0 could have the similar putative function with UGTPg100 to produce ginsenoside Rg₁/etc.Cluster-26044.27684 could have the similar putative function with Pn3-32-i5（Xyl）and Pn3-31 to produce notoginsenoside R1 and Rd/etc.Transcription factors were excavated by transcriptomics and their target gene/protein was verified to reveal the regulatory mechanism on ginsenoside biosynthesis.Transcriptomics and phylogenetic tree analysis showed that Pg WRKY4X is a novel WRKY transcription factor in P.ginseng.Subcellular localization assay indicated that Pg WRKY4X located in the nucleus.Correlation analysis found that there was a significant correlation between Pg WRKY4X and upstream ginsenoside biosynthesis genes,3-hydroxy-3-methylglutaryl-Co A reductase（HMGR）and squalene epoxidase（Pg SE）.Electrophoretic mobility shift assay and glutathione S-transferase pull-down assay showed Pg WRKY4X had interaction with Pg SE.Overexpression of Pg WRKY4X in transgenic cells of P.ginseng significantly enhanced ginsenosides accumulation by comprehensively upregulating ginsenosides biosynthetic genes,especially Pg SE.It indicated that Pg WRKY4X positively regulated ginsenosides biosynthesis via Pg SE as a possible target.Based on the above results of regulation mechanism,a plant synthesis system to produce ginsenoside Rg₃ by a combination modular strategy of metabolic engineering and chemical transformation was constructed.First,SE and Pq3-O-UGT2 were determined as the key enzymes for ginsenoside Rg₃ biosynthesis.The semi-rational design and site mutagenesis was performed to optimize the Pq3-O-UGT2 mutation withhighcatalytic efficiency.Second,CRISPR/Cas9 was used to knock down CYP716A53v2 expression inthe branch pathway of protopanaxtriol-type ginsenoside biosynthesis for enhancing the metabolic flux of protopanaxadiol-type ginsenoside Rg₃.Third,ginsenoside Rg₃ was accumulated in xylem which mainly consists of lignin as reported.Phenylalanine ammonia lyase（PAL）is a key lignin biosynthesis gene.Thus,PAL was overexpressed and it could improve ginsenoside Rg₃accumulation by accelerating xylem expansion.Then,combination the co-expression of ginsenoside aglycon synthetic gene,SE,Pq3-O-UGT2 mutation,PAL and CRISPR/Cas9 system was performed to improve ginsenoside Rg₃ accumulation.Finally,jasmonate stimulation and chemical transformation further increased the production of ginsenoside Rg₃ to 83.64 mg/L in shake flask（6.97±0.13mg/g,～200-fold higher than native wild-type and 2.58-fold higher than engineering microorganism）.In summary,this study provides a theoretical basis for the production of ginsenosides and notoginsenosides on plant chassis,and also provides a reference for the biotechnology production of other active ingredients...